Conjugates and compositions for cellular delivery

ABSTRACT

This invention features conjugates, compositions, methods of synthesis, and applications thereof, including folate derived conjugates of nucleosides, nucleotides, non-nucleosides, and nucleic acids including enzymatic nucleic acids and antisense nucleic acid molecules.

This patent application is a divisional of U.S. Ser. No. 10/151,116,filed May 17, 2002 which claims priority from U.S. Ser. No. 60/362,016,filed Mar. 6, 2002 and from U.S. Ser. No. 60/292,217, filed May 18,2001, both entitled ‘CONJUGATES AND COMPOSITIONS FOR CELLULAR DELIVERY’.These applications are hereby incorporated by reference herein in theirentirety including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing3USCNT3,”created on Aug. 31, 2010, which is 6,269 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to conjugates, compositions, methods ofsynthesis, and applications thereof. The discussion is provided only forunderstanding of the invention that follows. This summary is not anadmission that any of the work described below is prior art to theclaimed invention.

The cellular delivery of various therapeutic compounds, such asantiviral and chemotherapeutic agents, is usually compromised by twolimitations. First the selectivity of chemotherapeutic agents is oftenlow, resulting in high toxicity to normal tissues. Secondly, thetrafficking of many compounds into living cells is highly restricted bythe complex membrane systems of the cell. Specific transporters allowthe selective entry of nutrients or regulatory molecules, whileexcluding most exogenous molecules such as nucleic acids and proteins.Various strategies can be used to improve transport of compounds intocells, including the use of lipid carriers and various conjugatesystems. Conjugates are often selected based on the ability of certainmolecules to be selectively transported into specific cells, for examplevia receptor mediated endocytosis. By attaching a compound of interestto molecules that are actively transported across the cellularmembranes, the effective transfer of that compound into cells orspecific cellular organelles can be realized. Alternately, moleculesthat are able to penetrate cellular membranes without active transportmechanisms, for example, various lipophilic molecules, can be used todeliver compounds of interest. Examples of molecules that can beutilized as conjugates include but are not limited to peptides,hormones, fatty acids, vitamins, flavonoids, sugars, reporter molecules,reporter enzymes, chelators, porphyrins, intercalcators, and othermolecules that are capable of penetrating cellular membranes, either byactive transport or passive transport.

The delivery of compounds to specific cell types, for example, cancercells, can be accomplished by utilizing receptors associated withspecific cell types. Particular receptors are overexpressed in certaincancerous cells, including the high affinity folic acid receptor. Forexample, the high affinity folate receptor is a tumor marker that isoverexpressed in a variety of neoplastic tissues, including breast,ovarian, cervical, colorectal, renal, and nasoparyngeal tumors, but isexpressed to a very limited extent in normal tissues. The use of folicacid based conjugates to transport exogenous compounds across cellmembranes can provide a targeted delivery approach to the treatment anddiagnosis of disease and can provide a reduction in the required dose oftherapeutic compounds. Furthermore, therapeutic bioavialability,pharmacodynamics, and pharmacokinetic parameters can be modulatedthrough the use of bioconjugates, including folate bioconjugates. Godwinet al., 1972, J. Biol. Chem., 247, 2266-2271, report the synthesis ofbiologically active pteroyloligo-L-glutamates. Habus et al., 1998,Bioconjugate Chem., 9, 283-291, describe a method for the solid phasesynthesis of certain oligonucleotide-folate conjugates. Cook, U.S. Pat.No. 6,721,208, describes certain oligonucleotides modified with specificconjugate groups. The use of biotin and folate conjugates to enhancetransmembrane transport of exogenous molecules, including specificoligonucleotides has been reported by Low et al., U.S. Pat. Nos.5,416,016, 5,108,921, and International PCT publication No. WO 90/12096.Manoharan et al., International PCT publication No. WO 99/66063 describecertain folate conjugates, including specific nucleic acid folateconjugates with a phosphoramidite moiety attached to the nucleic acidcomponent of the conjugate, and methods for the synthesis of thesefolate conjugates. Nomura et al., 2000, J. Org. Chem., 65, 5016-5021,describe the synthesis of an intermediate,alpha-[2-(trimethylsilyl)ethoxycarbonl]folic acid, useful in thesynthesis of certain types of folate-nucleoside conjugates. Guzaev etal., U.S. Pat. No. 6,335,434, describes the synthesis of certain folateoligonucleotide conjugates.

SUMMARY OF THE INVENTION

The present invention features a compound having the formula I:

wherein each R₁, R₃, R₄, R₅, R₆, R₇ and R₈ is independently hydrogen,alkyl, substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a phosphorus containing group, nucleoside,nucleotide, small molecule, nucleic acid, or a solid support comprisinga linker.

The present invention features a compound having the formula II:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, nucleic acid, or a solid support comprising a linker.

The present invention features a compound having the formula III:

wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, or nucleic acid.

The present invention features a compound having the formula IV:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₂ is a phosphoruscontaining group, nucleoside, nucleotide, small molecule, nucleic acid,or a solid support comprising a linker, and R₁₃ is an amino acid sidechain.

The present invention features a compound having the formula V:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆, R₇ and R₈ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

The present invention features a compound having the formula VI:

wherein each R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, R₂ isa phosphorus containing group, nucleoside, nucleotide, small molecule,nucleic acid, or a solid support comprising a linker, each “n” isindependently an integer from 0 to about 200, and L is a degradablelinker.

The present invention features a compound having the formula VII:

wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, nucleic acid, or a solid support comprising a linker.

The present invention features a compound having the formula VIII:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

The present invention features a method for synthesizing a compoundhaving Formula V:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group,comprising: coupling a bis-hydroxy aminoalkyl derivative, for exampleD-threoninol, with a N-protected aminoalkanoic acid to yield a compoundof Formula IX;

wherein R₁₁ is an amino protecting group, R₁₂ is a straight or branchedchain alkyl, substituted alkyl, aryl, or substituted aryl, and each “n”is independently an integer from 0 to about 200; introducing primaryhydroxy protection R₁ followed by amino deprotection of R₁₁ to yield acompound of Formula X;

wherein R₁ is a protecting group, R₁₂ is a straight or branched chainalkyl, substituted alkyl, aryl, or substituted aryl, and each “n” isindependently an integer from 0 to about 200; coupling the deprotectedamine of Formula X with a protected amino acid, for example glutamicacid, to yield a compound of Formula XI;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each “n” is independently an integer from 0 to about 200, R₁₁ is anamino protecting group, and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl; deprotecting the amine R₁₁of the conjugated glutamic acid of Formula XI to yield a compound ofFormula XII;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each “n” is independently an integer from 0 to about 200, R₁₁ is anamino protecting group, and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl; coupling the deprotectedamine of Formula XII with an amino protected pteroic acid to yield acompound of Formula XIII;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each “n” is independently aninteger from 0 to about 200; and introducing a phosphorus containinggroup at the secondary hydroxyl of Formula XIII to yield a compound ofFormula V.

The present invention features a method for synthesizing a compoundhaving Formula VIII:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, each R₉ and R₁₀ is independently a nitrogen containing group,cyanoalkoxy, alkoxy, aryloxy, or alkyl group, and R₁₂ is a straight orbranched chain alkyl, substituted alkyl, aryl, or substituted aryl,comprising; coupling a bis-hydroxy aminoalkyl derivative, for exampleD-threoninol, with a protected amino acid, for example glutamic acid, toyield a compound of Formula XIV;

wherein R₁₁ is an amino protecting group, each “n” is independently aninteger from 0 to about 200, R₄ is independently a protecting group, andR₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl; introducing primary hydroxy protection R₁ followed byamino deprotection of R₁₁ of Formula XIV to yield a compound of FormulaXV;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and each “n” is independently an integer from 0 toabout 200; coupling the deprotected amine of Formula XV with an aminoprotected pteroic acid to yield a compound of Formula XVI;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each “n” is independently aninteger from 0 to about 200; and introducing a phosphorus containinggroup at the secondary hydroxyl of Formula XVI to yield a compound ofFormula VIII.

In one embodiment, R₂ of a compound of the invention comprises aphosphorus containing group.

In another embodiment, R₂ of a compound of the invention comprises anucleoside, for example, a nucleoside with beneficial activity such asanticancer or antiviral activity.

In yet another embodiment, R₂ of a compound of the invention comprises anucleotide, for example, a nucleotide with beneficial activity such asanticancer or antiviral activity.

In a further embodiment, R₂ of a compound of the invention comprises asmall molecule, for example, a small molecule with beneficial activitysuch as anticancer or antiviral activity.

In another embodiment, R₂ of a compound of the invention comprises anucleic acid, for example, a nucleic acid with beneficial activity suchas anticancer or antiviral activity.

In one embodiment, R₂ of a compound of the invention comprises a solidsupport comprising a linker.

In another embodiment, a nucleoside (R₂) of the invention comprises anucleoside with anticancer activity.

In another embodiment, a nucleoside (R₂) of the invention comprises anucleoside with antiviral activity.

In another embodiment, the nucleoside (R₂) of the invention comprisesfludarabine, lamivudine (3TC), 5-fluoro uridine, AZT, ara-adenosine,ara-adenosine monophosphate, a dideoxy nucleoside analog,carbodeoxyguanosine, ribavirin, fialuridine, lobucavir, a pyrophosphatenucleoside analog, an acyclic nucleoside analog, acyclovir,gangciclovir, penciclovir, famciclovir, an L-nucleoside analog, FTC,L-FMAU, L-ddC, L-FddC, L-d4C, L-Fd4C, an L-dideoxypurine nucleosideanalog, cytallene, bis-POM PMEA (GS-840), BMS-200,475, carbovir orabacavir.

In one embodiment, R₁₃ of a compound of the invention comprises analkylamino or an alkoxy group, for example, —CH₂O— or —CH(CH₂)CH₂O—.

In another embodiment, R₁₂ of a compound of the invention is analkylhyrdroxyl, for example, —(CH₂)_(n)OH, where n comprises an integerfrom about 1 to about 10.

In another embodiment, L of Formula VI of the invention comprisesserine, threonine, or a photolabile linkage.

In one embodiment, R₉ of a compound of the invention comprises aphosphorus protecting group, for example —OCH₂CH₂CN (oxyethylcyano).

In one embodiment, R₁₀ of a compound of the invention comprises anitrogen containing group, for example, —N(R₁₄) wherein R₁₄ is astraight or branched chain alkyl having from about 1 to about 10carbons.

In another embodiment, R₁₀ of a compound of the invention comprises aheterocycloalkyl or heterocycloalkenyl ring containing from about 4 toabout 7 atoms, and having from about 1 to about 3 heteroatoms comprisingoxygen, nitrogen, or sulfur.

In another embodiment, R₁ of a compound of the invention comprises anacid labile protecting group, such as a trityl or substituted tritylgroup, for example, a dimethoxytrityl or mono-methoxytrityl group.

In another embodiment, R₄ of a compound of the invention comprises atert-butyl, Fm (fluorenyl-methoxy), or allyl group.

In one embodiment, R₆ of a compound of the invention comprises a TFA(trifluoracetyl) group.

In another embodiment, R₃, R₅, R₇ and R₈ of a compound of the inventionare independently hydrogen.

In one embodiment, R₇ of a compound of the invention is independentlyisobutyryl, dimethylformamide, or hydrogen.

In another embodiment, R₁₂ of a compound of the invention comprises amethyl group or ethyl group.

In one embodiment, a nucleic acid of the invention comprises anenzymatic nucleic acid, for example a hammerhead, Inozyme, DNAzyme,G-cleaver, Zinzyme, Amberzyme, or allozyme.

In another embodiment, a nucleic acid of the invention comprises anantisense nucleic acid, 2-5 A nucleic acid chimera, or decoy nucleicacid.

In another embodiment, the solid support having a linker of theinvention comprises a structure of Formula XVII:

wherein SS is a solid support, and each “n” is independently an integerfrom about 1 to about 200.

In another embodiment, the solid support of the instant invention iscontrolled pore glass (CPG) or polystyrene, and can be used in thesynthesis of a nucleic acid.

In one embodiment, the invention features a pharmaceutical compositioncomprising a compound of the invention and a pharmaceutically acceptablecarrier.

In another embodiment, the invention features a method of treating acancer patient, comprising contacting cells of the patient with apharmaceutical composition of the invention under conditions suitablefor the treatment. This treatment can comprise the use of one or moreother drug therapies under conditions suitable for the treatment. Thecancers contemplated by the instant invention include but are notlimited to breast cancer, lung cancer, colorectal cancer, brain cancer,esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer,cervical cancer, head and neck cancer, ovarian cancer, melanoma,lymphoma, glioma, or multidrug resistant cancers.

In one embodiment, the invention features a method of treating a patientinfected with a virus, comprising contacting cells of the patient with apharmaceutical composition of the invention, under conditions suitablefor the treatment. This treatment can comprise the use of one or moreother drug therapies under conditions suitable for the treatment. Theviruses contemplated by the instant invention include but are notlimited to HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza,rhinovirus, west nile virus, Ebola virus, foot and mouth virus, andpapilloma virus.

In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule or other target molecule in asample, for example, a gene in a cancer cell, comprising a compound ofthe instant invention.

In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule, or other target molecule in asample, for example, a gene in a virus-infected cell, comprising acompound of the instant invention.

In another embodiment, the invention features a compound of the instantinvention comprising a modified phosphate group, for example, aphosphoramidite, phosphodiester, phosphoramidate, phosphorothioate,phosphorodithioate, alkylphosphonate, arylphosphonate, monophosphate,diphosphate, triphosphate, or pyrophosphate.

In one embodiment, the invention features a method for synthesizing acompound having Formula XVIII:

wherein each R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, comprising: reacting folic acid with acarboxypeptidase to yield a compound of Formula XIX;

introducing a protecting group R₆ on the secondary amine of Formula XIXto yield a compound of Formula XX;

wherein R₆ is a nitrogen protecting group; and introducing a protectinggroup R₇ on the primary amine of Formula XX to yield a compound ofFormula XVIII.

In another embodiment, the amino protected pteroic acid of the inventionis a compound of Formula XVIII.

In one embodiment, the invention encompasses a compound of Formula Ihaving Formula XXI:

wherein each “n” is independently an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of FormulaVII having Formula XXII:

wherein each “n” is independently an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of FormulaIV having Formula XXIII:

wherein “n” is an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of FormulaIV having Formula XXIV:

wherein “n” is an integer from 0 to about 200.

In another embodiment, the invention features a compound having FormulaXXV:

wherein each R₅ and R₇ is independently hydrogen, alkyl or a nitrogenprotecting group, each R₁₅, R₁₆, R₁₇, and R₁₈ is independently O, S,alkyl, substituted alkyl, aryl, substituted aryl, or halogen, X₁ is—CH(X_(1′)) or a group of Formula XXVI:

wherein R₄ is a protecting group and “n” is an integer from 0 to about200;

X_(1′) is the protected or unprotected side chain of a naturallyoccurring or non-naturally-occurring amino acid, X₂ is amide, alkyl, orcarbonyl containing linker or a bond, and X₃ is a degradable linkerwhich is optionally absent.

In another embodiment, the X₃ group of Formula XXV comprises a group ofFormula XXVI:

wherein R₄ is hydrogen or a protecting group, “n” is an integer from 0to about 200 and R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl.

In yet another embodiment, R₄ of Formula XXVI is hydrogen and R₁₂ ismethyl or hydrogen.

In still another embodiment, the invention features a compound havingFormula XXVII:

wherein “n” is an integer from about 0 to about 20, R₄ is H or acationic salt, and R₂₄ is a sulfurcontaining leaving group, for examplea group comprising:

In another embodiment, the invention features a method for synthesizinga compound having Formula XXVII comprising:

(a) selective tritylation of the thiol of cysteamine under conditionssuitable to yield a compound having Formula XXVIII:

wherein “n” is an integer from about 0 to about 20 and R₁₉ is a thiolprotecting group;

(b) peptide coupling of the product of (a) with a compound havingFormula XXIX:

wherein R₂₀ is a carboxylic acid protecting group and R₂₁ is an aminoprotecting group, under conditions suitable to yield a compound havingFormula XXX:

wherein “n” is an integer from about 0 to about 20, R₁₉ is a thiolprotecting group, R₂₀ is a carboxylic acid protecting group and R₂₁ isan amino protecting group;

(c) removing the amino protecting group R₂₁ of the product of (b) underconditions suitable to yield a compound having Formula XXXI:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b);

(d) condensation of the product of (c) with a compound having FormulaXXXII:

wherein R₂₂ is an amino protecting group, under conditions suitable toyield a compound having Formula XXXIII:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b) and R₂₂ is as described in (d);

(e) selective cleavage of R₂₂ from the product of (d) under conditionssuitable to yield a compound having Formula XXXIV:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b);

(f) coupling the product of (e) with a compound having Formula XXXV:

wherein R₂₃ is an amino protecting group under conditions suitable toyield a compound having Formula XXXVI:

wherein R₂₃ is an amino protecting group, “n” is an integer from about 0to about 20 and R₁₉ and R₂₀ are as described in (b);

(g) deprotecting the product of (f) under conditions suitable to yield acompound having Formula XXVIII.

wherein “n” is an integer from about 0 to about 20; and

(h) introducing a disulphide-based leaving group to the product of (g)under conditions suitable to yield a compound having Formula XXVII.

In one embodiment, the invention features a compound having FormulaXXIX:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide, and P is a phosphorus containinggroup.

In another embodiment, the invention features a method for synthesizinga compound having Formula XXIX, comprising:

(a) Coupling a thiol containing linker to a nucleic acid, polynucleotideor oligonucleotide under conditions suitable to yield a compound havingFormula XXX:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide, and P is a phosphorus containinggroup; and

(b) coupling the product of (a) with a compound having Formula XXVIIunder conditions suitable to yield a compound having Formula XXIX.

In another embodiment, the thiol containing linker of the invention is acompound having Formula XXXI:

wherein “n” is an integer from about 0 to about 20, P is a phosphoruscontaining group, for example a phosphine, phosphite, or phosphate, andR₂₄ is any alkyl, substituted alkyl, alkoxy, aryl, substituted aryl,alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group withor without additional protecting groups.

In another embodiment, the conditions suitable to yield a compoundhaving Formula XXX comprises reduction, for example using dithiothreitol(DTT) or any equivalent disulphide reducing agent, of the disulfide bondof a compound having Formula XXXII:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide, P is a phosphorus containing group,and R₂₄ is any alkyl, substituted alkyl, alkoxy, aryl, substituted aryl,alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group withor without additional protecting groups.

In one embodiment, the nucleic acid conjugates of the instant inventionare assembled post synthetically, for example, following solid phaseoligonucleotide synthesis.

The present invention provides compositions and conjugates comprisingnucleosidic and non-nucleosidic folate derivatives. The presentinvention also provides nucleic acid folate derivatives including RNA,DNA, and PNA based conjugates. The attachment of folate compounds of theinvention to nucleosides, nucleotides, non-nucleosides, and nucleic acidmolecules is provided at any position within the molecule, for example,at internucleotide linkages, nucleosidic sugar hydroxyl groups such as5′, 3′, and 2′-hydroxyls, and/or at nucleobase positions such as aminoand carbonyl groups.

The exemplary folate conjugates of the invention are described ascompounds of Formulae I-XXV, however, other folate and antifolatederivatives are provided by the invention, including various folateanalogs of the compounds of Formulae I-XXV, including dihydrofloates,tetrahydrofolates, tetrahydrorpterins, folinic acid, pteropolyglutamicacid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacids. As used herein, the term “folate” is meant to refer to folate andfolate derivatives, including pteroic acid derivatives and analogs.

The present invention features compositions and conjugates to facilitatedelivery of molecules into a biological system such as cells. The folateconjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosmall molecules, lipids, nucleosides, nucleotides, nucleic acids,negatively charged polymers and other polymers, for example proteins,peptides, carbohydrates, or polyamines. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system. The compounds of the invention generally shownin Formulae I-XXV, are expected to improve delivery of molecules into anumber of cell types originating from different tissues, in the presenceor absence of serum.

In one embodiment, the present invention features molecules,compositions and conjugates of molecules, for example, non-nucleosidicsmall molecules, nucleosides, nucleotides, and nucleic acids, such asenzymatic nucleic acid molecules, antisense nucleic acids, 2-5 Aantisense chimeras, triplex DNA, decoy RNA, aptamers, and antisensenucleic acids containing RNA cleaving chemical groups.

In another embodiment, the present invention features methods tomodulate gene expression, for example, genes involved in the progressionand/or maintenance of cancer or in a viral infection. For example, inone embodiment, the invention features the use of one or more of thenucleic acid-based molecules and methods independently or in combinationto inhibit the expression of the gene(s) encoding proteins associatedwith cancerous conditions, for example breast cancer, lung cancer,colorectal cancer, brain cancer, esophageal cancer, stomach cancer,bladder cancer, pancreatic cancer, cervical cancer, head and neckcancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrugresistant cancer associated genes.

In another embodiment, the invention features the use of one or more ofthe nucleic acid-based molecules and methods independently or incombination to inhibit the expression of the gene(s) encoding viralproteins, for example HIV, HBV, HCV, CMV, RSV, HSV, poliovirus,influenza, rhinovirus, west nile virus, Ebola virus, foot and mouthvirus, and papilloma virus associated genes.

In one embodiment, the invention features the use of an enzymaticnucleic acid molecule folate conjugate, preferably in the hammerhead,NCH, G-cleaver, amberzyme, zinzyme and/or DNAzyme motif, to inhibit theexpression of cancer and virus associated genes.

In another embodiment, the invention features the use of an enzymaticnucleic acid molecule as a folate conjugate. These enzymatic nucleicacids can catalyze the hydrolysis of RNA phosphodiester bonds in trans(and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseenzymatic nucleic acids. Without being bound by any particular theory,in general, enzymatic nucleic acids act by first binding to a targetRNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA destroys its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets. Thus, a single enzymaticnucleic acid molecule is able to cleave many molecules of target RNA. Inaddition, the enzymatic nucleic acid is a highly specific inhibitor ofgene expression, with the specificity of inhibition depending not onlyon the base-pairing mechanism of binding to the target RNA, but also onthe mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of an enzymatic nucleic acid.

In one embodiment of the invention described herein, the enzymaticnucleic acid molecule component of the folate conjugate is formed in ahammerhead or hairpin motif, but can also be formed in the motif of ahepatitis delta virus, group I intron, group II intron or RNase P RNA(in association with an RNA guide sequence), Neurospora VS RNA,DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of suchhammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992,AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampelet al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929,Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene,82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira &McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motifis described by Perrotta and Been, 1992 Biochemistry 31, 16; of theRNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster andAltman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res.24, 835; Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363);Group H introns are described by Griffin et al., 1995, Chem. Biol. 2,761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al.,International PCT Publication No. WO 96/22689; of the Group I intron byCech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al.,International PCT Publication No. WO 95/11304; Chartrand et al., 1995,NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al.,1997, PNAS 94, 4262, and Beigelman et al., International PCT publicationNo. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat,International PCT Publication No. WO 98/58058; and G-cleavers aredescribed in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 andEckstein et al., International PCT Publication No. WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993),Amberzyme (Class I motif; FIG. 3; Beigelman et al., U.S. Ser. No.09/301,511) and Zinzyme (FIG. 4) (Beigelman et al., U.S. Ser. No.09/301,511), all incorporated by reference herein including drawings,can also be used in the present invention. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule (Cech et al.,U.S. Pat. No. 4,987,071).

In one embodiment of the present invention, a nucleic acid moleculecomponent of a conjugate of the instant invention can be between 12 and100 nucleotides in length. For example, enzymatic nucleic acid moleculesof the invention are preferably between 15 and 50 nucleotides in length,more preferably between 25 and 40 nucleotides in length, e.g., 34, 36,or 38 nucleotides in length (for example see Jarvis et al., 1996, J.Biol. Chem., 271, 29107-29112). Exemplary DNAzymes of the invention arepreferably between 15 and 40 nucleotides in length, more preferablybetween 25 and 35 nucleotides in length, e.g., 29, 30, 31, or 32nucleotides in length (see for example Santoro et al., 1998,Biochemistry, 37, 13330-13342; Chartrand et al., 1995, Nucleic AcidsResearch, 23, 4092-4096). Exemplary antisense molecules of the inventionare preferably between 15 and 75 nucleotides in length, more preferablybetween 20 and 35 nucleotides in length, e.g., 25, 26, 27, or 28nucleotides in length (see, for example, Woolf et al., 1992, PNAS., 89,7305-7309; Milner et al., 1997, Nature Biotechnology, 15, 537-541).Exemplary triplex forming oligonucleotide molecules of the invention arepreferably between 10 and 40 nucleotides in length, more preferablybetween 12 and 25 nucleotides in length, e.g., 18, 19, 20, or 21nucleotides in length (see for example Maher et al., 1990, Biochemistry,29, 8820-8826; Strobel and Dervan, 1990, Science, 249, 73-75). Thoseskilled in the art will recognize that all that is required is for thenucleic acid molecule to be of sufficient length and suitableconformation for the nucleic acid molecule to catalyze a reactioncontemplated herein. The length of the nucleic acid molecules describedand exemplified herein are not not limiting within the general sizeranges stated.

The folate conjugates of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The conjugates and/or conjugatecomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection or infusion pump, with or without theirincorporation in biopolymers. The compositions and conjugates of theinstant invention, individually, or in combination or in conjunctionwith other drugs, can be used to treat diseases or conditions discussedabove. For example, to treat a disease or condition associated with thelevels of a pathogenic protein, the patient can be treated, or otherappropriate cells can be treated, as is evident to those skilled in theart, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the described molecules can be used incombination with other known treatments to treat conditions or diseasesdiscussed above. For example, the described molecules can be used incombination with one or more known therapeutic agents to treat breast,lung, prostate, colorectal, brain, esophageal, bladder, pancreatic,cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma,multidrug resistant cancers, and/or HIV, HBV, HCV, CMV, RSV, HSV,poliovirus, influenza, rhinovirus, west nile virus, Ebola virus, footand mouth virus, and papilloma virus infection.

Included in another embodiment are a series of multi-domain cellulartransport vehicles (MCTV) including one or more compounds of FormulaI-XXV that enhance the cellular uptake and transmembrane permeability ofnegatively charged molecules in a variety of cell types. The compoundsof the invention are used either alone or in combination with othercompounds with a neutral or a negative charge including but not limitedto neutral lipid and/or targeting components, to improve theeffectiveness of the formulation or conjugate in delivering andtargeting the predetermined compound or molecule to cells. Anotherembodiment of the invention encompasses the utility of these compoundsfor increasing the transport of other impermeable and/or lipophiliccompounds into cells. Targeting components include ligands for cellsurface receptors including, peptides and proteins, glycolipids, lipids,carbohydrates, and their synthetic variants, for example folatereceptors.

In another embodiment, the compounds of the invention are provided as asurface component of a lipid aggregate, such as a liposome encapsulatedwith the predetermined molecule to be delivered. Liposomes, which can beunilamellar or multilamellar, can introduce encapsulated material into acell by different mechanisms. For example, the liposome can directlyintroduce its encapsulated material into the cell cytoplasm by fusingwith the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) and itscontents released from the liposome and out of the acidic vacuole intothe cellular cytoplasm.

In one embodiment the invention features a lipid aggregate formulationof Formulae I-XXV, including phosphatidylcholine (of varying chainlength; e.g., egg yolk phosphatidylcholine), cholesterol, a cationiclipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000(DSPE-PEG2000). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In another embodimentthis cationic lipid aggregate comprises a covalently bound compounddescribed in any of the Formula I-XXV.

In another embodiment, polyethylene glycol (PEG) is covalently attachedto the compounds of the present invention. The attached PEG can be anymolecular weight but is preferably between 2000-50,000 daltons.

The compounds and methods of the present invention are useful forintroducing nucleotides, nucleosides, nucleic acid molecules, lipids,peptides, proteins, and/or non-nucleosidic small molecules into a cell.For example, the invention can be used for nucleotide, nucleoside,nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic smallmolecule delivery where the corresponding target site of action existsintracellularly.

In one embodiment, the compounds of the instant invention provideconjugates of molecules that can interact with folate receptors, such ashigh affinity folate receptors, and provide a number of features thatallow the efficient delivery and subsequent release of conjugatedcompounds across biological membranes. The compounds utilize chemicallinkages between the folate and the compound to be delivered of lengththat can interact preferentially with folate receptors. Furthermore, thechemical linkages between the folate and the compound to be deliveredcan be designed as degradable linkages, for example by utilizing aphosphate linkage that is proximal to a nucleophile, such as a hydroxylgroup. Deprotonation of the hydroxyl group or an equivalent group, as aresult of pH or interaction with a nuclease, can result in nucleophilicattack of the phosphate resulting in a cyclic phosphate intermediatethat can be hydrolyzed. This cleavage mechanism is analogous RNAcleavage in the presence of a base or RNA nuclease. Alternately, otherdegradable linkages can be selected that respond to various factors suchas UV irradiation, cellular nucleases, pH, temperature etc. The use ofdegradable linkages allows the delivered compound to be released in apredetermined system, for example in the cytoplasm of a cell, or in aparticular cellular organelle.

The present invention also provides folate derived phosphoramidites thatare readily conjugated to compounds and molecules of interest.Phosphoramidite compounds of the invention permit the direct attachmentof folate conjugates to molecules of interest without the need for usingnucleic acid phosphoramidite species as scaffolds. As such, the used ofphosphoramidite chemistry can be used directly in coupling the folateconjugates to a compound of interest, without the need for othercondensation reactions, such as condensation of the folate to an aminogroup on the nucleic acid, for example at the N6 position of adenosineor a 2′-deoxy-2′-amino function. Additionally, compounds of theinvention can be used to introduce non-nucleic acid based folateconjugated linkages into oligonucleotides that can provide moreefficient coupling during oligonucleotide synthesis than the use ofnucleic acid-based folate phosphoramidites. This improved coupling cantake into account improved steric considerations of abasic ornon-nucleosidic scaffolds bearing pendant alkyl linkages.

Compounds of the invention utilizing triphosphate groups can be utilizedin the enzymatic incorporation of conjugate molecules intooligonucleotides. Such enzymatic incorporation is useful when conjugatesare used in post-synthetic enzymatic conjugation or selection reactions,(see for example Matulic-Adamic et al., 2000, Bioorg. Med. Chem. Lett.,10, 1299-1302; Lee et al., 2001, NAR., 29, 1565-1573; Joyce, 1989, Gene,82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992,Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268;Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17,89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra;Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish etal., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr. Opin.Chem. Biol., 4, 669).

Compounds of the invention can be used to detect the presence of atarget molecule in a biological system, such as tissue, cell or celllysate. Examples of target molecules include nucleic acids, proteins,peptides, antibodies, polysaccharides, lipids, hormones, sugars, metals,microbial or cellular metabolites, analytes, pharmaceuticals, and otherorganic and inorganic molecules or other biomolecules in a sample. Thecompounds of the instant invention can be conjugated to a predeterminedcompound or molecule that is capable of interacting with the targetmolecule in the system and providing a detectable signal or response.Various compounds and molecules known in the art that can be used inthese applications include but are not limited to antibodies, labeledantibodies, allozymes, aptamers, labeled nucleic acid probes, molecularbeacons, fluorescent molecules, radioisotopes, polysaccharides, and anyother compound capable of interacting with the target molecule andgenerating a detectable signal upon target interaction. For example,such compounds are described in Application entitled “NUCLEIC ACIDSENSOR MOLECULES” filed on Mar. 6, 2001 Ser. No. 09/800,594 withinventors Nassim Usman and James A. McSwiggen, which is incorporated byreference in its entirety, including the drawings.

The term “target molecule” as used herein, refers to nucleic acidmolecules, proteins, peptides, antibodies, polysaccharides, lipids,sugars, metals, microbial or cellular metabolites, analytes,pharmaceuticals, and other organic and inorganic molecules that arepresent in a system.

By “inhibit” or “down-regulate” it is meant that the expression of thegene, or level of RNAs or equivalent RNAs encoding one or more proteinsubunits, or activity of one or more protein subunits, such aspathogenic protein, viral protein or cancer related protein subunit(s),is reduced below that observed in the absence of the compounds orcombination of compounds of the invention. In one embodiment, inhibitionor down-regulation with an enzymatic nucleic acid molecule preferably isbelow that level observed in the presence of an enzymatically inactiveor attenuated molecule that is able to bind to the same site on thetarget RNA, but is unable to cleave that RNA. In another embodiment,inhibition or down-regulation with antisense oligonucleotides ispreferably below that level observed in the presence of, for example, anoligonucleotide with scrambled sequence or with mismatches. In anotherembodiment, inhibition or down-regulation of viral or oncogenic RNA,protein, or protein subunits with a compound of the instant invention isgreater in the presence of the compound than in its absence.

By “up-regulate” is meant that the expression of the gene, or level ofRNAs or equivalent RNAs encoding one or more protein subunits, oractivity of one or more protein subunits, such as viral or oncogenicprotein subunit(s), is greater than that observed in the absence of thecompounds or combination of compounds of the invention. For example, theexpression of a gene, such as a viral or cancer related gene, can beincreased in order to treat, prevent, ameliorate, or modulate apathological condition caused or exacerbated by an absence or low levelof gene expression.

By “modulate” is meant that the expression of the gene, or level of RNAsor equivalent RNAs encoding one or more protein subunits, or activity ofone or more protein subunit(s) of a protein, for example a viral orcancer related protein is up-regulated or down-regulated, such that theexpression, level, or activity is greater than or less than thatobserved in the absence of the compounds or combination of compounds ofthe invention.

The term “enzymatic nucleic acid molecule” as used herein refers to anucleic acid molecule which has complementarity in a substrate bindingregion to a specified gene target, and also has an enzymatic activitywhich is active to specifically cleave target RNA. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave RNAand thereby inactivate a target RNA molecule. These complementaryregions allow sufficient hybridization of the enzymatic nucleic acidmolecule to the target RNA and thus permit cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% canalso be useful in this invention (see for example Werner and Uhlenbeck,1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids canbe modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving and/or ligation activity to the molecule (Cech et al., U.S.Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

The term “nucleic acid molecule” as used herein, refers to a moleculehaving nucleotides. The nucleic acid can be single, double, or multiplestranded and can comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof.

The term “enzymatic portion” or “catalytic domain” as used herein refersto that portion/region of the enzymatic nucleic acid molecule essentialfor cleavage of a nucleic acid substrate (for example see FIG. 1).

The term “substrate binding arm” or “substrate binding domain” as usedherein refers to that portion/region of a enzymatic nucleic acid whichis able to interact, for example via complementarity (i.e., able tobase-pair with), with a portion of its substrate. Preferably, suchcomplementarity is 100%, but can be less if desired. For example, as fewas 10 bases out of 14 can be base-paired (see for example Werner andUhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al.,1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of sucharms are shown generally in FIGS. 1-4. That is, these arms containsequences within a enzymatic nucleic acid which are intended to bringenzymatic nucleic acid and target RNA together through complementarybase-pairing interactions. The enzymatic nucleic acid of the inventioncan have binding arms that are contiguous or non-contiguous and can beof varying lengths. The length of the binding arm(s) are preferablygreater than or equal to four nucleotides and of sufficient length tostably interact with the target RNA; preferably 12-100 nucleotides; morepreferably 14-24 nucleotides long (see for example Werner and Uhlenbeck,supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranceet al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, thedesign is such that the length of the binding arms are symmetrical(i.e., each of the binding arms is of the same length; e.g., five andfive nucleotides, or six and six nucleotides, or seven and sevennucleotides long) or asymmetrical (i.e., the binding arms are ofdifferent length; e.g., six and three nucleotides; three and sixnucleotides long; four and five nucleotides long; four and sixnucleotides long; four and seven nucleotides long; and the like).

The term “Inozyme” or “NCH” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described asNCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNAsubstrates having a cleavage triplet NCH/, where N is a nucleotide, C iscytidine and H is adenosine, uridine or cytidine, and/represents thecleavage site. H is used interchangeably with X. Inozymes can alsopossess endonuclease activity to cleave RNA substrates having a cleavagetriplet NCN/, where N is a nucleotide, C is cytidine, and/represents thecleavage site. “I” in FIG. 2 represents an Inosine nucleotide,preferably a ribo-Inosine or xylo-Inosine nucleoside.

The term “G-cleaver” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described asG-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity tocleave RNA substrates having a cleavage triplet NYN/, where N is anucleotide, Y is uridine or cytidine and/represents the cleavage site.G-cleavers can be chemically modified as is generally shown in FIG. 2.

The term “amberzyme” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described inFIG. 2. Amberzymes possess endonuclease activity to cleave RNAsubstrates having a cleavage triplet NG/N, where N is a nucleotide, G isguanosine, and/represents the cleavage site. Amberzymes can bechemically modified to increase nuclease stability through substitutionsas are generally shown in FIG. 3. In addition, differing nucleosideand/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′loops shown in the figure. Amberzymes represent a non-limiting exampleof an enzymatic nucleic acid molecule that does not require aribonucleotide (2′-OH) group within its own nucleic acid sequence foractivity.

The term “zinzyme” motif as used herein, refers to an enzymatic nucleicacid molecule comprising a motif as is generally described in FIG. 3.Zinzymes possess endonuclease activity to cleave RNA substrates having acleavage triplet including but not limited to YG/Y, where Y is uridineor cytidine, and G is guanosine and/represents the cleavage site.Zinzymes can be chemically modified to increase nuclease stabilitythrough substitutions as are generally shown in FIG. 3, includingsubstituting 2′-O-methyl guanosine nucleotides for guanosinenucleotides. In addition, differing nucleotide and/or non-nucleotidelinkers can be used to substitute the 5′-gaaa-2′ loop shown in thefigure. Zinzymes represent a non-limiting example of an enzymaticnucleic acid molecule that does not require a ribonucleotide (2′-OH)group within its own nucleic acid sequence for activity.

The term ‘DNAzyme’ as used herein, refers to an enzymatic nucleic acidmolecule that does not require the presence of a 2′-OH group for itsactivity. In particular embodiments the enzymatic nucleic acid moleculecan have an attached linker(s) or other attached or associated groups,moieties, or chains containing one or more nucleotides with 2′-OHgroups. DNAzymes can be synthesized chemically or expressed endogenouslyin vivo, by means of a single stranded DNA vector or equivalent thereof.An example of a DNAzyme is shown in FIG. 4 and is generally reviewed inUsman et al., International PCT Publication No. WO 95/11304; Chartrandet al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655;Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, NatureBiotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem.Soc., 122, 2433-39. Additional DNAzyme motifs can be selected for usingtechniques similar to those described in these references, and hence,are within the scope of the present invention.

The term “sufficient length” as used herein, refers to anoligonucleotide of length great enough to provide the intended functionunder the expected condition, i.e., greater than or equal to 3nucleotides. For example, for binding arms of enzymatic nucleic acid“sufficient length” means that the binding arm sequence is long enoughto provide stable binding to a target site under the expected bindingconditions. Preferably, the binding arms are not so long as to preventuseful turnover of the nucleic acid molecule.

The term “stably interact” as used herein, refers to interaction of theoligonucleotides with target nucleic acid (e.g., by forming hydrogenbonds with complementary nucleotides in the target under physiologicalconditions) that is sufficient to the intended purpose (e.g., cleavageof target RNA by an enzyme).

The term “homology” as used herein, refers to the nucleotide sequence oftwo or more nucleic acid molecules is partially or completely identical.

The term “antisense nucleic acid”, as used herein, refers to anon-enzymatic nucleic acid molecule that binds to target RNA by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the target RNA(for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf etal., U.S. Pat. No. 5,849,902). Typically, antisense molecules arecomplementary to a target sequence along a single contiguous sequence ofthe antisense molecule. However, in certain embodiments, an antisensemolecule can bind to substrate such that the substrate molecule forms aloop, and/or an antisense molecule can bind such that the antisensemolecule forms a loop. Thus, the antisense molecule can be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. For a review of currentantisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274,21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al.,1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol.,313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke,1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be usedto target RNA by means of DNA-RNA interactions, thereby activating RNaseH, which digests the target RNA in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region,which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof.

The term “RNase H activating region” as used herein, refers to a region(generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target RNA to form a non-covalent complex thatis recognized by cellular RNase H enzyme (see for example Arrow et al.,U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). TheRNase H enzyme binds to the nucleic acid molecule-target RNA complex andcleaves the target RNA sequence. The RNase H activating regioncomprises, for example, phosphodiester, phosphorothioate (preferably atleast four of the nucleotides are phosphorothiote substitutions; morespecifically, 4-11 of the nucleotides are phosphorothiotesubstitutions); phosphorodithioate, 5′-thiophosphate, ormethylphosphonate backbone chemistry or a combination thereof. Inaddition to one or more backbone chemistries described above, the RNaseH activating region can also comprise a variety of sugar chemistries.For example, the RNase H activating region can comprise deoxyribose,arabino, fluoroarabino or a combination thereof, nucleotide sugarchemistry. Those skilled in the art will recognize that the foregoingare non-limiting examples and that any combination of phosphate, sugarand base chemistry of a nucleic acid that supports the activity of RNaseH enzyme is within the scope of the definition of the RNase H activatingregion and the instant invention.

The term “2-5 A antisense chimera” as used herein, refers to anantisense oligonucleotide containing a 5′-phosphorylated 2′-5′-linkedadenylate residue. These chimeras bind to target RNA in asequence-specific manner and activate a cellular 2-5 A-dependentribonuclease which, in turn, cleaves the target RNA (Torrence et al.,1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000,Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol.Ther., 78, 55-113).

The term “triplex forming oligonucleotides” as used herein, refers to anoligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al.,2000, Biochim. Biophys. Acta, 1489, 181-206).

The term “gene” it as used herein, refers to a nucleic acid that encodesan RNA, for example, nucleic acid sequences including but not limited tostructural genes encoding a polypeptide.

The term “pathogenic protein” as used herein, refers to endogenous orexongenous proteins that are associated with a disease state orcondition, for example a particular cancer or viral infection.

The term “complementarity” refers to the ability of a nucleic acid toform hydrogen bond(s) with another RNA sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., enzymatic nucleic acid cleavage, antisense or triplehelix inhibition. Determination of binding free energies for nucleicacid molecules is well known in the art (see, e.g., Turner et al., 1987,CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat.Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule which can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%,90%, and 100% complementary). “Perfectly complementary” means that allthe contiguous residues of a nucleic acid sequence will hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence.

The term “RNA” as used herein, refers to a molecule comprising at leastone ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant anucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety.

The term “decoy RNA” as used herein, refers to a RNA molecule or aptamerthat is designed to preferentially bind to a predetermined ligand. Suchbinding can result in the inhibition or activation of a target molecule.The decoy RNA or aptamer can compete with a naturally occurring bindingtarget for the binding of a specific ligand. For example, it has beenshown that over-expression of HIV trans-activation response (TAR) RNAcan act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specificexample and those in the art will recognize that other embodiments canbe readily generated using techniques generally known in the art, seefor example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody andGold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2,100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000,Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a receptor and blockthe binding of an effector molecule or a decoy RNA can be designed tobind to receptor of interest and prevent interaction with the receptor.

The term “cell” as used herein, refers to its usual biological sense,and does not refer to an entire multicellular organism. The cell can,for example, be in vitro, e.g., in cell culture, or present in amulticellular organism, including, e.g., birds, plants and mammals suchas humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cellcan be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalianor plant cell).

The term “highly conserved sequence region” as used herein, refers to anucleotide sequence of one or more regions in a target gene does notvary significantly from one generation to the other or from onebiological system to the other.

The term “non-nucleotide” as used herein, refers to any group orcompound which can be incorporated into a nucleic acid chain in theplace of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound is abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine.

The term “nucleotide” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are recognized in the art to include natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of chemically modified and other natural nucleicacid bases that can be introduced into nucleic acids include, forexample, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

The term “nucleoside” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a sugar. Nucleosides arerecognized in the art to include natural bases (standard), and modifiedbases well known in the art. Such bases are generally located at the 1′position of a nucleoside sugar moiety. Nucleosides generally comprise abase and sugar group. The nucleosides can be unmodified or modified atthe sugar, and/or base moiety, (also referred to interchangeably asnucleoside analogs, modified nucleosides, non-natural nucleosides,non-standard nucleosides and other; see for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra all are hereby incorporated by reference herein).There are several examples of modified nucleic acid bases known in theart as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.Some of the non-limiting examples of chemically modified and othernatural nucleic acid bases that can be introduced into nucleic acidsinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhdroxymethyl)uridine,5-(carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleoside bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

The term “cap structure” as used herein, refers to chemicalmodifications, which have been incorporated at either terminus of theoligonucleotide (see for example Wincott et al., WO 97/26270,incorporated by reference herein). These terminal modifications protectthe nucleic acid molecule from exonuclease degradation, and can help indelivery and/or localization within a cell. The cap can be present atthe 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can bepresent on both terminus. In non-limiting examples, the 5′-cap includesinverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

The term “abasic” as used herein, refers to sugar moieties lacking abase or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative (for more details see Wincott et al., International PCTpublication No. WO 97/26270).

The term “unmodified nucleoside” as used herein, refers to one of thebases adenine, cytosine, guanine, thymine, uracil joined to the 1′carbon of β-D-ribo-furanose.

The term “modified nucleoside” as used herein, refers to any nucleotidebase which contains a modification in the chemical structure of anunmodified nucleotide base, sugar and/or phosphate.

The term “consists essentially of” as used herein, is meant that theactive nucleic acid molecule of the invention, for example, an enzymaticnucleic acid molecule, contains an enzymatic center or core equivalentto those in the examples, and binding arms able to bind RNA such thatcleavage at the target site occurs. Other sequences can be present whichdo not interfere with such cleavage. Thus, a core region can, forexample, include one or more loop, stem-loop structure, or linker whichdoes not prevent enzymatic activity. For example, a core sequence for ahammerhead enzymatic nucleic acid can comprise a conserved sequence,such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is5′-GCCGUUAGGC-3′ (SEQ ID NO 1), or any other Stem II region known in theart, or a nucleotide and/or non-nucleotide linker. Similarly, for othernucleic acid molecules of the instant invention, such as Inozyme,G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5 A antisense,triplex forming nucleic acid, and decoy nucleic acids, other sequencesor non-nucleotide linkers can be present that do not interfere with thefunction of the nucleic acid molecule.

Sequence X can be a linker of ≧2 nucleotides in length, preferably 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferablybe internally base-paired to form a stem of preferably ≧2 base pairs. Inyet another embodiment, the nucleotide linker X can be a nucleic acidaptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer(TAR) and others (for a review see Gold et al., 1995, Annu. Rev.Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed.Gesteland and Atkins, pp. 511, CSH Laboratory Press). A “nucleic acidaptamer” as used herein is meant to indicate a nucleic acid sequencecapable of interacting with a ligand. The ligand can be any natural or asynthetic molecule, including but not limited to a resin, metabolites,nucleosides, nucleotides, drugs, toxins, transition state analogs,peptides, lipids, proteins, amino acids, nucleic acid molecules,hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

Alternatively or in addition, sequence X can be a non-nucleotide linker.Non-nucleotides can include abasic nucleotide, polyether, polyamine,polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.Specific examples include those described by Seela and Kaiser, NucleicAcids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload andSchepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz,J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic AcidsRes. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.,Biochemistry 1991, 30:9914; Arnold et al., International Publication No.WO 89/02439; Usman et al., International Publication No. WO 95/06731;Dudycz et al., International Publication No. WO 95/11910 and Ferentz andVerdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated byreference herein. A “non-nucleotide” further means any group or compoundwhich can be incorporated into a nucleic acid chain in the place of oneor more nucleotide units, including either sugar and/or phosphatesubstitutions, and allows the remaining bases to exhibit their enzymaticactivity. The group or compound can be abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment,the invention features an enzymatic nucleic acid molecule having one ormore non-nucleotide moieties, and having enzymatic activity to cleave anRNA or DNA molecule.

The term “patient” as used herein, refers to an organism, which is adonor or recipient of explanted cells or the cells themselves. “Patient”also refers to an organism to which the nucleic acid molecules of theinvention can be administered. Preferably, a patient is a mammal ormammalian cells. More preferably, a patient is a human or human cells.

The term “enhanced enzymatic activity” as used herein, includes activitymeasured in cells and/or in vivo where the activity is a reflection ofboth the catalytic activity and the stability of the nucleic acidmolecules of the invention. In this invention, the product of theseproperties can be increased in vivo compared to an all RNA enzymaticnucleic acid or all DNA enzyme. In some cases, the activity or stabilityof the nucleic acid molecule can be decreased (i.e., less thanten-fold), but the overall activity of the nucleic acid molecule isenhanced, in vivo.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and can or can not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements can be present.

The term “negatively charged molecules” as used herein, refers tomolecules such as nucleic acid molecules (e.g., RNA, DNA,oligonucleotides, mixed polymers, peptide nucleic acid, and the like),peptides (e.g., polyaminoacids, polypeptides, proteins and the like),nucleotides, pharmaceutical and biological compositions, that havenegatively charged groups that can ion-pair with the positively chargedhead group of the cationic lipids of the invention.

The term “coupling” as used herein, refers to a reaction, eitherchemical or enzymatic, in which one atom, moiety, group, compound ormolecule is joined to another atom, moiety, group, compound or molecule.

The terms “deprotection” or “deprotecting” as used herein, refers to theremoval of a protecting group.

The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain “isoalkyl”, andcyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio,alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom about 1 to about 7 carbons, more preferably about 1 to about 4carbons. The alkyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” alsoincludes alkenyl groups containing at least one carbon-carbon doublebond, including straight-chain, branched-chain, and cyclic groups.Preferably, the alkenyl group has about 2 to about 12 carbons. Morepreferably it is a lower alkenyl of from about 2 to about 7 carbons,more preferably about 2 to about 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl,alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substitutedaryl groups. The term “alkyl” also includes alkynyl groups containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group hasabout 2 to about 12 carbons. More preferably it is a lower alkynyl offrom about 2 to about 7 carbons, more preferably about 2 to about 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moietiesof the invention can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. The preferred substituent(s)of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano,alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” grouprefers to an alkyl group (as described above) covalently joined to anaryl group (as described above). Carbocyclic aryl groups are groupswherein the ring atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted. Heterocyclic aryl groups aregroups having from about 1 to about 3 heteroatoms as ring atoms in thearomatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, methoxyethyl or ethoxymethyl.

The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkylthioether, for example, methylthiomethyl or methylthioethyl.

The term “amino” as used herein refers to a nitrogen containing group asis known in the art derived from ammonia by the replacement of one ormore hydrogen radicals by organic radicals. For example, the terms“aminoacyl” and “aminoalkyl” refer to specific N-substituted organicradicals with acyl and alkyl substituent groups respectively.

The term “amination” as used herein refers to a process in which anamino group or substituted amine is introduced into an organic molecule.

The term “exocyclic amine protecting moiety” as used herein refers to anucleobase amino protecting group compatible with oligonucleotidesynthesis, for example, an acyl or amide group.

The term “alkenyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl,and 2-methyl-3-heptene.

The term “alkoxy” as used herein refers to an alkyl group of indicatednumber of carbon atoms attached to the parent molecular moiety throughan oxygen bridge. Examples of alkoxy groups include, for example,methoxy, ethoxy, propoxy and isopropoxy.

The term “alkynyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon triple bond. Examples of “alkynyl” include propargyl,propyne, and 3-hexyne.

The term “aryl” as used herein refers to an aromatic hydrocarbon ringsystem containing at least one aromatic ring. The aromatic ring canoptionally be fused or otherwise attached to other aromatic hydrocarbonrings or non-aromatic hydrocarbon rings. Examples of aryl groupsinclude, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthaleneand biphenyl. Preferred examples of aryl groups include phenyl andnaphthyl.

The term “cycloalkenyl” as used herein refers to a C3-C8 cyclichydrocarbon containing at least one carbon-carbon double bond. Examplesof cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “cycloalkyl” as used herein refers to a C3-C8 cyclichydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “cycloalkylalkyl,” as used herein, refers to a C3-C7 cycloalkylgroup attached to the parent molecular moiety through an alkyl group, asdefined above. Examples of cycloalkylalkyl groups includecyclopropylmethyl and cyclopentylethyl.

The terms “halogen” or “halo” as used herein refers to indicatefluorine, chlorine, bromine, and iodine.

The term “heterocycloalkyl,” as used herein refers to a non-aromaticring system containing at least one heteroatom selected from nitrogen,oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused toor otherwise attached to other heterocycloalkyl rings and/ornon-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups havefrom 3 to 7 members. Examples of heterocycloalkyl groups include, forexample, piperazine, morpholine, piperidine, tetrahydrofuran,pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups includepiperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

The term “heteroaryl” as used herein refers to an aromatic ring systemcontaining at least one heteroatom selected from nitrogen, oxygen, andsulfur. The heteroaryl ring can be fused or otherwise attached to one ormore heteroaryl rings, aromatic or non-aromatic hydrocarbon rings orheterocycloalkyl rings. Examples of heteroaryl groups include, forexample, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline andpyrimidine. Preferred examples of heteroaryl groups include thienyl,benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

The term “C1-C6 hydrocarbyl” as used herein refers to straight,branched, or cyclic alkyl groups having 1-6 carbon atoms, optionallycontaining one or more carbon-carbon double or triple bonds. Examples ofhydrocarbyl groups include, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl andpropargyl. When reference is made herein to C1-C6 hydrocarbyl containingone or two double or triple bonds it is understood that at least twocarbons are present in the alkyl for one double or triple bond, and atleast four carbons for two double or triple bonds.

The term “protecting group” as used herein, refers to groups known inthe art that are readily introduced and removed from an atom, forexample O, N, P, or S. Protecting groups are used to prevent undesirablereactions from taking place that can compete with the formation of aspecific compound or intermediate of interest. See also “ProtectiveGroups in Organic Synthesis”, 3rd Ed., 1999, Greene, T. W. and relatedpublications.

The term “nitrogen protecting group,” as used herein, refers to groupsknown in the art that are readily introduced on to and removed from anitrogen. Examples of nitrogen protecting groups include Boc, Cbz,benzoyl, and benzyl. See also “Protective Groups in Organic Synthesis”,3rd Ed., 1999, Greene, T. W. and related publications.

The term “hydroxy protecting group,” or “hydroxy protection” as usedherein, refers to groups known in the art that are readily introduced onto and removed from an oxygen, specifically an —OH group. Examples ofhyroxy protecting groups include trityl or substituted trityl goups,such as monomethoxytrityl and dimethoxytrityl, or substituted silylgroups, such as tert-butyldimethyl, trimethylsilyl, ortert-butyldiphenyl silyl groups. See also “Protective Groups in OrganicSynthesis”, 3rd Ed., 1999, Greene, T. W. and related publications.

The term “acyl” as used herein refers to —C(O)R groups, wherein R is analkyl or aryl.

The term “phosphorus containing group” as used herein, refers to achemical group containing a phosphorus atom. The phosphorus atom can betrivalent or pentavalent, and can be substituted with O, H, N, S, C orhalogen atoms. Examples of phosphorus containing groups of the instantinvention include but are not limited to phosphorus atoms substitutedwith O, H, N, S, C or halogen atoms, comprising phosphonate,alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramiditegroups, nucleotides and nucleic acid molecules.

The term “phosphine” or “phosphite” as used herein refers to a trivalentphosphorus species, for example compounds having Formula XXXII:

-   -   wherein R can include the groups:

-   -   and wherein S and T independently include the groups:

The term “phosphate” as used herein refers to a pentavalent phosphorusspecies, for example a compound having Formula XXXIV:

-   -   wherein R includes the groups:

-   -   and wherein S and T each independently can be a sulfur or oxygen        atom or a group which can include:

-   -   and wherein M comprises a sulfur or oxygen atom. The phosphate        of the invention can comprise a nucleotide phosphate, wherein        any R, S, or T in Formula XXXIV comprises a linkage to a nucleic        acid or nucleoside.

The term “cationic salt” as used herein refers to any organic orinorganic salt having a net positive charge, for example atriethylammonium (TEA) salt.

The term “degradable linker” as used herein, refers to linker moietiesthat are capable of cleavage under various conditions. Conditionssuitable for cleavage can include but are not limited to pH, UVirradiation, enzymatic activity, temperature, hydrolysis, elimination,and substitution reactions, and thermodynamic properties of the linkage.

The term “photolabile linker” as used herein, refers to linker moietiesas are known in the art, that are selectively cleaved under particularUV wavelengths. Compounds of the invention containing photolabilelinkers can be used to deliver compounds to a target cell or tissue ofinterest, and can be subsequently released in the presence of a UVsource.

The term “nucleic acid conjugates” as used herein, refers to nucleoside,nucleotide and oligonucleotide conjugates.

The term “folate” as used herein, refers to analogs and derivatives offolic acid, for example antifolates, dihydrofloates, tetrahydrofolates,tetrahydrorpterins, folinic acid, pteropolyglutamic acid, 1-deza,3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza,8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acidderivatives.

The term “compounds with neutral charge” as used herein, refers tocompositions which are neutral or uncharged at neutral or physiologicalpH. Examples of such compounds are cholesterol and other steroids,cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline,distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid,phosphatidic acid and its derivatives, phosphatidyl serine, polyethyleneglycol-conjugated phosphatidylamine, phosphatidylcholine,phosphatidylethanolamine and related variants, prenylated compoundsincluding farnesol, polyprenols, tocopherol, and their modified forms,diacylsuccinyl glycerols, fusogenic or pore forming peptides,dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

The term “lipid aggregate” as used herein refers to a lipid-containingcomposition wherein the lipid is in the form of a liposome, micelle(non-lamellar phase) or other aggregates with one or more lipids.

The term “biological system” as used herein, refers to a eukaryoticsystem or a prokaryotic system, can be a bacterial cell, plant cell or amammalian cell, or can be of plant origin, mammalian origin, yeastorigin, Drosophila origin, or archebacterial origin.

The term “systemic administration” as used herein refers to the in vivosystemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include, without limitations:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexpose the desired negatively charged polymers, e.g., nucleic acids, toan accessible diseased tissue. The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, incertain tissue types, such as the tissues of the reticular endothelialsystem (RES). A liposome formulation which can facilitate theassociation of drug with the surface of cells, such as, lymphocytes andmacrophages is also useful. This approach can provide enhanced deliveryof the drug to target cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of abnormal cells, such asthe cancer cells.

The term “pharmacological composition” or “pharmaceutical formulation”refers to a composition or formulation in a form suitable foradministration, for example, systemic administration, into a cell orpatient, preferably a human. Suitable forms, in part, depend upon theuse or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationto reach a target cell (i.e., a cell to which the negatively chargedpolymer is targeted).

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will be first described briefly.

DRAWINGS

FIG. 1 shows examples of chemically stabilized ribozyme motifs. HH Rz,represents hammerhead ribozyme motif (Usman et al., 1996, Curr. Op.Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig& Sproat, International PCT Publication No. WO 98/58058); G-Cleaver,represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic AcidsResearch 26, 4116-4120, Eckstein et al., International PCT publicationNo. WO 99/16871). N or n, represent independently a nucleotide which canbe same or different and have complementarity to each other; rI,represents ribo-Inosine nucleotide; arrow indicates the site of cleavagewithin the target. Position 4 of the HH Rz and the NCH Rz is shown ashaving 2′-C-allyl modification, but those skilled in the art willrecognize that this position can be modified with other modificationswell known in the art, so long as such modifications do notsignificantly inhibit the activity of the ribozyme.

FIG. 2 shows an example of the Amberzyme ribozyme motif that ischemically stabilized (see for example Beigelman et al., InternationalPCT publication No. WO 99/55857).

FIG. 3 shows an example of the Zinzyme A ribozyme motif that ischemically stabilized (see for example Beigelman et al., Beigelman etal., International PCT publication No. WO 99/55857).

FIG. 4 shows an example of a DNAzyme motif described by Santoro et al.,1997, PNAS, 94, 4262.

FIG. 5 shows a synthetic scheme for the synthesis of a folate conjugateof the instant invention.

FIG. 6 shows representative examples of fludarabine-folate conjugatemolecules of the invention.

FIG. 7 shows a synthetic scheme for post-synthetic modification of anucleic acid molecule to produce a folate conjugate.

FIG. 8 shows a synthetic scheme for generating a protected pteroic acidsynthon of the invention.

FIG. 9 shows a synthetic scheme for generating a 2-dithiopyridylactivated folic acid synthon of the invention.

FIG. 10 shows a synthetic scheme for generating an oligonucleotide ornucleic acid-folate conjugate.

FIG. 11 shows a an alternative synthetic scheme for generating anoligonucleotide or nucleic acid-folate conjugate.

FIG. 12 shows an alternative synthetic scheme for post-syntheticmodification of a nucleic acid molecule to produce a folate conjugate.

METHOD OF USE

The compositions and conjugates of the instant invention can be used toadminister pharmaceutical agents. Pharmaceutical agents prevent, inhibitthe occurrence, or treat (alleviate a symptom to some extent, preferablyall of the symptoms) of a disease state in a patient.

Generally, the compounds of the instant invention are introduced by anystandard means, with or without stabilizers, buffers, and the like, toform a pharmaceutical composition. For use of a liposome deliverymechanism, standard protocols for formation of liposomes can befollowed. The compositions of the present invention can also beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions; suspensions for injectable administration; and the like.

The present invention also includes pharmaceutically acceptableformulations of the compounds described above, preferably in combinationwith the molecule(s) to be delivered. These formulations include saltsof the above compounds, e.g., acid addition salts, for example, salts ofhydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

In one embodiment, the invention features the use of the compounds ofthe invention in a composition comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes). In another embodiment,the invention features the use of compounds of the invention covalentlyattached to polyethylene glycol. These formulations offer a method forincreasing the accumulation of drugs in target tissues. This class ofdrug carriers resists opsonization and elimination by the mononuclearphagocytic system (MPS or RES), thereby enabling longer bloodcirculation times and enhanced tissue exposure for the encapsulated drug(Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem.Pharm. Bull. 1995, 43, 1005-1011). Such compositions have been shown toaccumulate selectively in tumors, presumably by extravasation andcapture in the neovascularized target tissues (Lasic et al., Science1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,86-90). The long-circulating compositions enhance the pharmacokineticsand pharmacodynamics of therapeutic compounds, such as DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating compositions are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes a composition(s) prepared forstorage or administration that includes a pharmaceutically effectiveamount of the desired compound(s) in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. Forexample, preservatives, stabilizers, dyes and flavoring agents can beincluded in the composition. Examples of such agents include but are notlimited to sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. In addition, antioxidants and suspending agents can be included inthe composition.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors which those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer. Furthermore, the compounds ofthe invention and formulations thereof can be administered to a fetusvia administration to the mother of a fetus.

The compounds of the invention and formulations thereof can beadministered orally, topically, parenterally, by inhalation or spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a nucleic acid moleculeof the invention and a pharmaceutically acceptable carrier. One or morenucleic acid molecules of the invention can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients. Thepharmaceutical compositions containing nucleic acid molecules of theinvention can be in a form suitable for oral use, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example, sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds of the invention can also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Compounds of the invention can be administered parenterally in a sterilemedium. The drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anesthetics, preservatives and buffering agentscan be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The compounds of the present invention can also be administered to apatient in combination with other therapeutic compounds to increase theoverall therapeutic effect. The use of multiple compounds to treat anindication can increase the beneficial effects while reducing thepresence of side effects.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small refers to nucleic acid motifs less than about 100 nucleotides inlength, preferably less than about 80 nucleotides in length, and morepreferably less than about 50 nucleotides in length; e.g., antisenseoligonucleotides, hammerhead or the NCH ribozymes) are preferably usedfor exogenous delivery. The simple structure of these moleculesincreases the ability of the nucleic acid to invade targeted regions ofRNA structure. Exemplary molecules of the instant invention arechemically synthesized, and others can similarly be synthesized.

Oligonucleotides (eg; antisense GeneBlocs) are synthesized usingprotocols known in the art as described in Caruthers et al., 1992,Methods in Enzymology 211, 3-19, Thompson et al., International PCTPublication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res.23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennanet al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.6,001,311. All of these references are incorporated herein by reference.The synthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. In a non-limiting example, smallscale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer using a 0.2 mmol scale protocol with a 2.5 min coupling stepfor 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxynucleotides. Table II outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 mmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. In a non-limiting example, a 33-fold excess(60 μl of 0.11 M=6.6 mmol) of 2′-O-methyl phosphoramidite and a 105-foldexcess of S-ethyl tetrazole (60 μL of 0.25 M=15 mop can be used in eachcoupling cycle of 2′-O-methyl residues relative to polymer-bound5′-hydroxyl. In a non-limiting example, a 22-fold excess (40 μL of 0.11M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyltetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycleof deoxy residues relative to polymer-bound 5′-hydroxyl. Averagecoupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include but are not limited to;detritylation solution is 3% TCA in methylene chloride (ART); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the antisense oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aq. methylamine(1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatantis removed from the polymer support. The support is washed three timeswith 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is thenadded to the first supernatant. The combined supernatants, containingthe oligoribonucleotide, are dried to a white powder. Standard drying orlyophilization methods known to those skilled in the art can be used.

The method of synthesis used for normal RNA including certain enzymaticnucleic acid molecules follows the procedure as described in Usman etal., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, NucleicAcids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes useof common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 7.5 min coupling step for alkylsilyl protected nucleotides and a2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlinesthe amounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 mmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include; detritylation solution is3% TCA in methylene chloride (ABI); capping is performed with 16%N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mMpyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson SynthesisGrade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) isadded and the vial is heated at 65° C. for 15 min. The sample is cooledat −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 min. The cartridge is then washed again with water, salt exchangedwith 1 M NaCl and washed with water again. The oligonucleotide is theneluted with 30% acetonitrile.

Inactive hammerhead ribozymes or binding attenuated control ((BAC)oligonucleotides) are synthesized by substituting a U for G₅ and a U forA₁₄ (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20,3252). Similarly, one or more nucleotide substitutions can be introducedin other enzymatic nucleic acid molecules to inactivate the molecule andsuch molecules can serve as a negative control.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including, but notlimited to, 96 well format, with the ratio of chemicals used in thereaction being adjusted accordingly.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204).

The nucleic acid molecules of the present invention are modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). Ribozymes are purified by gelelectrophoresis using general methods or are purified by high pressureliquid chromatography (HPLC; See Wincott et al., Supra, the totality ofwhich is hereby incorporated herein by reference) and are re-suspendedin water.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) that prevent their degradation by serumribonucleases can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al.,supra; all of these describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules herein). Modifications which enhance their efficacy in cells,and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; allof the references are hereby incorporated in their totality by referenceherein). Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without inhibiting catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid molecules of the instant invention.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorothioate, and/or 5′-methylphosphonatelinkages improves stability, too many of these modifications may causesome toxicity. Therefore, when designing nucleic acid molecules theamount of these internucleotide linkages should be minimized. Withoutbeing bound by any particular theory, the reduction in the concentrationof these linkages should lower toxicity resulting in increased efficacyand higher specificity of these molecules.

Nucleic acid molecules having chemical modifications that maintain orenhance activity are provided. Such nucleic acid is also generally moreresistant to nucleases than unmodified nucleic acid. Thus, in a celland/or in vivo the activity can not be significantly lowered.Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acidmolecules and antisense nucleic acid molecules) delivered exogenouslyare optimally stable within cells until translation of the target RNAhas been inhibited long enough to reduce the levels of the undesirableprotein. This period of time varies between hours to days depending uponthe disease state. The nucleic acid molecules should be resistant tonucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of RNA and DNA (Wincottet al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,Methods in Enzymology 211, 3-19 (incorporated by reference herein) haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

Use of the nucleic acid-based molecules of the invention can lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple antisense or enzymatic nucleicacid molecules targeted to different genes, nucleic acid moleculescoupled with known small molecule inhibitors, or intermittent treatmentwith combinations of molecules (including different motifs) and/or otherchemical or biological molecules). The treatment of patients withnucleic acid molecules can also include combinations of different typesof nucleic acid molecules.

In another embodiment, nucleic acid catalysts having chemicalmodifications that maintain or enhance enzymatic activity are provided.Such nucleic acids are also generally more resistant to nucleases thanunmodified nucleic acid. Thus, in a cell and/or in vivo the activity ofthe nucleic acid can not be significantly lowered. As exemplified hereinsuch enzymatic nucleic acids are useful in a cell and/or in vivo even ifactivity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry,35, 14090). Such enzymatic nucleic acids herein are said to “maintain”the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.

In another aspect the nucleic acid molecules comprise a 5′ and/or a3′-cap structure.

In another embodiment the 3′-cap includes, for example 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

In one embodiment, the invention features modified enzymatic nucleicacid molecules with phosphate backbone modifications comprising one ormore phosphorothioate, phosphorodithioate, methylphosphonate,morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide,sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/oralkylsilyl, substitutions. For a review of oligonucleotide backbonemodifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues:Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, andMesmaeker et al., 1994, Novel Backbone Replacements forOligonucleotides, in Carbohydrate Modifications in Antisense Research,ACS, 24-39. These references are hereby incorporated by referenceherein.

In connection with 2′-modified nucleotides as described for theinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO98/28317, respectively, which are both incorporated by reference intheir entireties.

Various modifications to nucleic acid (e.g., antisense and ribozyme)structure can be made to enhance the utility of these molecules. Forexample, such modifications can enhance shelf-life, half-life in vitro,stability, and ease of introduction of such oligonucleotides to thetarget site, including e.g., enhancing penetration of cellular membranesand conferring the ability to recognize and bind to targeted cells.

Use of these molecules can lead to better treatment of diseaseprogression by affording the possibility of combination therapies (e.g.,multiple enzymatic nucleic acid molecules targeted to different genes,enzymatic nucleic acid molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations of enzymaticnucleic acid molecules (including different enzymatic nucleic acidmolecule motifs) and/or other chemical or biological molecules). Thetreatment of patients with nucleic acid molecules can also includecombinations of different types of nucleic acid molecules. Therapies canbe devised which include a mixture of enzymatic nucleic acid molecules(including different enzymatic nucleic acid molecule motifs), antisenseand/or 2-5 A chimera molecules to one or more targets to alleviatesymptoms of a disease.

Indications

Particular disease states that can be treated using compounds andcompositions of the invention include, but are not limited to, cancersand cancerous conditions such as breast, lung, prostate, colorectal,brain, esophageal, stomach, bladder, pancreatic, cervical, head andneck, and ovarian cancer, melanoma, lymphoma, glioma, multidrugresistant cancers, and/or viral infections including HIV, HBV, HCV, CMV,RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus, Ebolavirus, foot and mouth virus, and papilloma virus infection.

The molecules of the invention can be used in conjunction with otherknown methods, therapies, or drugs. For example, the use of monoclonalantibodies (eg; mAb IMC C225, mAB ABX-EGF) treatment, tyrosine kinaseinhibitors (TKIs), for example OSI-774 and ZD1839, chemotherapy, and/orradiation therapy, are all non-limiting examples of a methods that canbe combined with or used in conjunction with the compounds of theinstant invention. Common chemotherapies that can be combined withnucleic acid molecules of the instant invention include variouscombinations of cytotoxic drugs to kill the cancer cells. These drugsinclude, but are not limited to, paclitaxel (Taxol), docetaxel,cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracilcarboplatin, edatrexate, gemcitabine, vinorelbine etc. Those skilled inthe art will recognize that other drug compounds and therapies can besimilarly be readily combined with the compounds of the instantinvention are hence within the scope of the instant invention.

Diagnostic Uses

The compounds of this invention, for example, nucleic acid conjugatemolecules, can be used as diagnostic tools to examine genetic drift andmutations within diseased cells or to detect the presence of a diseaserelated RNA in a cell. The close relationship between, for example,enzymatic nucleic acid molecule activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple enzymatic nucleic acid moleculesconjugates of the invention, one can map nucleotide changes which areimportant to RNA structure and function in vitro, as well as in cellsand tissues. Cleavage of target RNAs with enzymatic nucleic acidmolecules can be used to inhibit gene expression and define the role(essentially) of specified gene products in the progression of disease.In this manner, other genetic targets can be defined as importantmediators of the disease. These experiments can lead to better treatmentof the disease progression by affording the possibility of combinationaltherapies (e.g., multiple enzymatic nucleic acid molecules targeted todifferent genes, enzymatic nucleic acid molecules coupled with knownsmall molecule inhibitors, or intermittent treatment with combinationsof enzymatic nucleic acid molecules and/or other chemical or biologicalmolecules). Other in vitro uses of enzymatic nucleic acid molecules ofthis invention are well known in the art, and include detection of thepresence of mRNAs associated with a disease-related condition. Such RNAis detected by determining the presence of a cleavage product aftertreatment with an enzymatic nucleic acid molecule using standardmethodology.

In a specific example, enzymatic nucleic acid molecules that aredelivered to cells as conjugates and which cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstenzymatic nucleic acid molecule is used to identify wild-type RNApresent in the sample and the second enzymatic nucleic acid molecule isused to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth enzymatic nucleic acid molecules to demonstrate the relativeenzymatic nucleic acid molecule efficiencies in the reactions and theabsence of cleavage of the “non-targeted” RNA species. The cleavageproducts from the synthetic substrates also serve to generate sizemarkers for the analysis of wild-type and mutant RNAs in the samplepopulation. Thus each analysis requires two enzymatic nucleic acidmolecules, two substrates and one unknown sample which is combined intosix reactions. The presence of cleavage products is determined using anRNAse protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype is adequate to establishrisk. If probes of comparable specific activity are used for bothtranscripts, then a qualitative comparison of RNA levels will beadequate and will decrease the cost of the initial diagnosis. Highermutant form to wild-type ratios are correlated with higher risk whetherRNA levels are compared qualitatively or quantitatively. The use ofenzymatic nucleic acid molecules in diagnostic applications contemplatedby the instant invention is more fully described in George et al., U.S.Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332,Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

Additional Uses

Potential uses of sequence-specific enzymatic nucleic acid molecules ofthe instant invention that are delivered to cells as conjugates can havemany of the same applications for the study of RNA that DNA restrictionendonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev.Biochem. 44:273). For example, the pattern of restriction fragments canbe used to establish sequence relationships between two related RNAs,and large RNAs can be specifically cleaved to fragments of a size moreuseful for study. The ability to engineer sequence specificity of theenzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknownsequence. Applicant has described the use of nucleic acid molecules todown-regulate gene expression of target genes in bacterial, microbial,fungal, viral, and eukaryotic systems including plant, or mammaliancells.

EXAMPLE 1 Synthesis ofO¹-(4-monomethoxytrityl)-N-(6-(N-(α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroic acid conjugate3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20) (FIG. 5)

General. All reactions were carried out under a positive pressure ofargon in anhydrous solvents. Commercially available reagents andanhydrous solvents were used without further purification. ¹H (400.035MHz) and ³¹P (161.947 MHz) NMR spectra were recorded in CDCl₃, unlessstated otherwise, and chemical shifts in ppm refer to TMS and H₃PO₄,respectively. Analytical thin-layer chromatography (TLC) was performedwith Merck Art. 5554 Kieselgel 60 F₂₅₄ plates and flash columnchromatography using Merck 0.040-0.063 mm silica gel 60.

N-(N-Fmoc-6-aminocaproyl)-D-threoninol (13). N-Fmoc-6-aminocaproic acid(10 g, 28.30 mmol) was dissolved in DMF (50 ml) and N-hydroxysuccinimide(3.26 g, 28.30 mmol) and 1,3-dicyclohexylcarbodiimide (5.84 g, 28.3mmol) were added to the solution. The reaction mixture was stirred at RT(about 23° C.) overnight and the precipitated 1,3-dicyclohexylureafiltered off. To the filtrate D-threoninol (2.98 g, 28.30 mmol) wasadded and the reaction mixture stirred at RT overnight. The solution wasreduced to ca half the volume in vacuo, the residue diluted with about mml of ethyl acetate and extracted with about x ml of 5% NaHCO₃, followedby washing with brine. The organic layer was dried (Na₂SO₄), evaporatedto a syrup and chromatographed by silica gel column chromatography using1-10% gradient of methanol in ethyl acetate. Fractions containing theproduct were pooled and evaporated to a white solid (9.94 g, 80%).¹H-NMR (DMSO-d₆-D₂O)

7.97-7.30 (m, 8H, aromatic), 4.34 (d, J=6.80, 2H, Fm), 4.26 (t, J=6.80,1H, Fm), 3.9 (m, 1H, H3 Thr), 3.69 (m, 1H, H2 Thr), 3.49 (dd, J=10.6,J=7.0, 1H, H1 Thr), 3.35 (dd, J=10.6, J=6.2, 1H, H1′ Thr), 3.01 (m, 2H,CH₂CO Acp), 2.17 (m, 2H, CH₂NH Acp), 1.54 (m, 2H, CH₂ Acp), 1.45 (m, 2H,CH₂ Acp), 1.27 (m, 2H, CH₂ Acp), 1.04 (d, J=6.4, 3H, CH₃). MS/ESI⁺ m/z441.0 (M+H)⁺.

O¹-(4-Monomethoxytrityl)-N-(N-Fmoc-6-aminocaproyl)-D-threoninol (14). Tothe solution of 13 (6 g, 13.62 mmol) in dry pyridine (80 ml)p-anisylchlorodiphenyl-methane (6 g, 19.43 mmol) was added and thereaction mixture stirred at RT overnight. Methanol was added (20 ml) andthe solution concentrated in vacuo. The residual syrup was partitionedbetween about x ml of dichloromethane and about x ml of 5% NaHCO₃, theorganic layer was washed with brine, dried (Na₂SO₄) and evaporated todryness. Flash column chromatography using 1-3% gradient of methanol indichloromethane afforded 14 as a white foam (6 g, 62%). ¹H-NMR (DMSO)

7.97-6.94 (m, 22H, aromatic), 4.58 (d, 1H, J=5.2, OH), 4.35 (d, J=6.8,2H, Fm), 4.27 (t, J=6.8, 1H, Fm), 3.97 (m, 2H, H2, H3 Thr), 3.80 (s, 3H,OCH₃), 3.13 (dd, J=8.4, J=5.6, 1H, H1 Thr), 3.01 (m, 2H, CH₂CO Acp),2.92 (m, dd, J=8.4, J=6.4, 1H, H1′ Thr), 2.21 (m, 2H, CH₂NH Acp), 1.57(m, 2H, CH₂ Acp), 1.46 (m, 2H, CH₂ Acp), 1.30 (m, 2H, CH₂ Acp), 1.02 (d,J=5.6, 3H, CH₃). MS/ESI⁺ m/z 735.5 (M+Na)⁺.

O¹-(4-Monomethoxytrityl)-N-(6-aminocaproyl)-D-threoninol (15). 14 (9.1g, 12.77 mmol) was dissolved in DMF (100 ml) containing piperidine (10ml) and the reaction mixture was kept at RT for about 1 hour. Thesolvents were removed in vacuo and the residue purified by silica gelcolumn chromatography using 1-10% gradient of methanol indichloromethane to afford 15 as a syrup (4.46 g, 71%). ¹H-NMR

7.48-6.92 (m, 14H, aromatic), 6.16 (d, J=8.8, 1H, NH), 4.17 (m, 1H, H3Thr), 4.02 (m, 1H, H2 Thr), 3.86 (s, 3H, OCH₃), 3.50 (dd, J=9.7, J=4.4,1H, H1 Thr), 3.37 (dd, J=9.7, J=3.4, 1H, H1′ Thr), 2.78 (t, J=6.8, 2H,CH₂CO Acp), 2.33 (t, J=7.6, 2H, CH₂NH Acp), 1.76 (m, 2H, CH₂ Acp), 1.56(m, 2H, CH₂ Acp), 1.50 (m, 2H, CH₂ Acp), 1.21 (d, J=6.4, 3H, CH₃).MS/ESI⁺ m/z 491.5 (M+H)⁺.

O¹-(4-Monomethoxytrityl)-N-(6-(N-(N-Boc-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol(16). To the solution of N-Boc-α-OFm-glutamic acid (Bachem) (1.91 g,4.48 mmol) in DMF (10 ml) N-hydroxysuccinimide (518 mg, 4.50 mmol) and1,3-dicyclohexylcarbodiimide (928 mg, 4.50 mmol) was added and thereaction mixture was stirred at RT overnight. 1,3-Dicyclohexylurea wasfiltered off and to the filtrate 15 (2 g, 4.08 mmol) and pyridine (2 ml)were added. The reaction mixture was stirred at RT for 3 hours and thanconcentrated in vacuo. The residue was partitioned between ethyl acetateand 5% Na₂HCO₃, the organic layer extracted with brine as previouslydescribed, dried (Na₂SO₄) and evaporated to a syrup. Columnchromatography using 2-10% gradient of methanol in dichlotomethaneafforded 16 as a white foam (3.4 g, 93%). ¹H-NMR

7.86-6.91 (m, 22H, aromatic), 6.13 (d, J=8.8, 1H, NH), 5.93 (br s, 1H,NH), 5.43 (d, J=8.4, 1H, NH), 4.63 (dd, J=10.6, J=6.4, 1H, Fm), 4.54(dd, J=10.6, J=6.4, 1H, Fm), 4.38 (m, 1H, Glu), 4.3 (t, J=6.4, 1H, Fm),4.18 (m, 1H, H3 Thr), 4.01 (m, 1H, H2 Thr), 3.88 (s, 3H, OCH₃), 3.49(dd, J=9.5, J=4.4, 1H, H1 Thr), 3.37 (dd, J=9.5, J=3.8, 1H, H1′ Thr),3.32 (m, 2H, CH₂CO Acp), 3.09 (br s, 1H, OH), 2.32 (m, 2H, CH₂NH Acp),2.17 (m, 3H, Glu), 1.97 (m, 1H, Glu), 1.77 (m, 2H, CH₂ Acp), 1.61 (m,2H, CH₂ Acp), 1.52 (s, 9H, t-Bu), 1.21 (d, J=6.4, 3H, CH₃). MS/ESI⁺ m/z920.5 (M+Na)⁺.

N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol hydrochloride (17).16 (2 g, 2.23 mmol) was dissolved in methanol (30 ml) containing anisole(10 ml) and to this solution x ml of 4M HCl in dioxane was added. Thereaction mixture was stirred for 3 hours at RT and then concentrated invacuo. The residue was dissolved in ethanol and the product precipitatedby addition of x ml of ether. The precipitate was washed with ether anddried to give 17 as a colorless foam (1 g, 80%). ¹H-NMR (DMSO-d₆-D₂O)

7.97-7.40 (m, 8H, aromatic), 4.70 (m, 1H, Fm), 4.55 (m, 1H, Fm), 4.40(t, J=6.4, 1H, Fm), 4.14 (t, J=6.6, 1H, Glu), 3.90 (dd, J=2.8, J=6.4,1H, H3 Thr), 3.68 (m, 1H, H2 Thr), 3.49 (dd, J=10.6, J=7.0, 1H, H1 Thr),3.36 (dd, J=10.6, J=6.2, 1H, H1′ Thr), 3.07 (m, 2H, CH₂CO Acp), 2.17 m,3H), 1.93 (m, 2H), 1.45 (m, 2H), 1.27 (m, 2H), 1.04 (d, J=6.4, 3H Thr).MS/ESI⁺ m/z 526.5 (M+H)⁺.

N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (18). To the solution of N²-iBu-N¹⁰-TFA-pteroic acid¹(480 mg, 1 mmol) in DMF (5 ml) 1-hydroxybenzotriazole (203 mg, 1.50mmol), EDCI (288 mg, 1.50 mmol) and 17 (free base, 631 mg, 1.2 mmol) areadded. The reaction mixture is stirred at RT for 2 hours, thenconcentrated to ca 3 ml and loaded on the column of silica gel. Elutionwith dichloromethane, followed by 1-20% gradient of methanol indichloromethane afforded 18 (0.5 g, 51%). ¹H-NMR (DMSO-d₆-D₂O) δ 9.09(d, J=6.8, 1H, NH) 8.96 (s, 1H, H7 pteroic acid), 8.02-7.19 (m, 13H,aromatic, NH), 5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.41 (d,J=6.8, 2H, Fm), 4.29 (t, J=6.8, 1H, Fm), 3.89 (dd, J=6.2, J=2.8, 1H, H3Thr), 3.68 (m, 1H, H2 Thr), 3.48 (dd, J=10.4, J=7.0, 1H, H1 Thr), 3.36(dd, J=10.4, J=6.2, 1H H1′ Thr), 3.06 (m, 2H, CH₂CO Acp), 2.84 (m, 1H,iBu), 2.25 (m, 2H, CH₂NH Acp), 2.16 (m, 3H, Glu), 1.99 (m, 1H, Glu),1.52 (m, 2H Acp), 1.42 (m, 2H Acp), 1.27 (m, 2H Acp), 1.20 (s, 3H iBu),1.19 (s, 3H, iBu), 1.03 (d, J=6.2, 3H Thr). MS/ESI⁻ m/z 984.5 (M−H)⁻.

O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (19). To the solution of conjugate 18 (1 g, 1.01 mmol) indry pyridine (15 ml) p-anisylchlorodiphenylmethane (405 mg) was addedand the reaction mixture was stirred, protected from moisture, at RTovernight. Methanol (3 ml) was added and the reaction mixtureconcentrated to a syrup in vacuo. The residue was partitioned betweendichloromethane and 5% NaHCO₃, the organic layer washed with brine,dried (Na₂SO₄) and evaporated to dryness. Column chromatography using0.5-10% gradient of methanol in dichloromethane afforded 19 as acolorless foam (0.5 g, 39%. ¹H-NMR (DMSO-d₆-D₂O δ9.09 (d, J=6.8, 1H, NH)8.94 (s, 1H, H7 pteroic acid), 8.00-6.93 (m, 27H, aromatic, NH), 5.30(s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.40 (d, J=6.8, 2H, Fm), 4.29(t, J=6.8, 1H, Fm), 3.94 (m, 2H, H3, H2 Thr), 3.79 (s, 3H, OCH₃) 3.11(dd, J=8.6, J=5.8, 1H, H1 Thr), 3.04 (m, 2H, CH₂CO Acp), 2.91 (dd,J=8.6, J=6.4, 1H, H1′ Thr), 2.85 (m, 1H, iBu), 2.25 (m, 2H, CH₂NH Acp),2.19 (m, 2H, Glu), 2.13 (m, 1H, Glu), 1.98 (m, 1H, Glu), 1.55 (m, 2HAcp), 1.42 (m, 2H Acp), 1.29 (m, 211 Acp), 1.20 (s, 3H iBu), 1.18 (s,3H, iBu), 1.00 (d, J=6.4, 3H Thr). MS/ESI⁻ m/z 1257.0 (M−H)⁻.

O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl)-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate 3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20).To the solution of 19 (500 mg, 0.40 mmol) in dichloromethane (2 ml)2-cyanoethyl tetraisopropylphosphordiamidite (152 μL, 0.48 mmol) wasadded followed by pyridinium trifluoroacetate (93 mg, 0.48 mmol). Thereaction mixture was stirred at RT for 1 hour and than loaded on thecolumn of silica gel in hexanes. Elution using ethyl acetate-hexanes1:1, followed by ethyl acetate and ethyl acetate-acetone 1:1 in thepresence of 1% pyridine afforded 20 as a colorless foam (480 mg, 83%).³¹P NMR δ 149.4 (s), 149.0 (s).

EXAMPLE 2 Synthesis of 2-dithiopyridyl activated folic acid (30) (FIG.9)

Synthesis of the cysteamine modified folate 30 is presented in FIG. 9.Monomethoxytrityl cysteamine 21 was prepared by selective tritylation ofthe thiol group of cysteamine with 4-methoxytrityl alcohol intrifluoroacetic acid. Peptide coupling of 21 with Fmoc-Glu-OtBu (BachemBioscience Inc., King of Prussia, Pa.) in the presence of PyBOP yielded22 in a high yield. N-Fmoc group was removed smoothly with piperidine togive 23. Condensation of 23 with p-(4-methoxytrityl)aminobenzoic acid,prepared by reaction of p-aminobenzoic acid with 4-methoxytritylchloride in pyridine, afforded the fully protected conjugate 24.Selective cleavage of N-MMTr group with acetic acid afforded 25 inquantitative yield. Shiff base formation between 25 andN²-iBu-6-formylpterin 26,⁹ followed by reduction with borane-pyridinecomplex proceeded with a good yield to give fully protectedcysteamine-folate adduct 27.¹² The consecutive cleavage of protectinggroups of 27 with base and acid yielded thiol derivative 29. The thiolexchange reaction of 29 with 2,2-dipyridyl disulfide afforded thedesired S-pyridyl activated synthon 30 as a yellow powder; Isolated as aTEA⁺ salt: ¹H NMR spectrum for 10 in D₂O: δ 8.68 (s, 1H, H-7), 8.10 (d,J=3.6, 1H, pyr), 7.61 (d, J=8.8, 2H, PABA), 7.43 (m, 1H, pyr), 7.04 (d,J=7.6, 1H, pyr), 6.93 (m, 1H, pyr), 6.82 (d, J=8.8, 1H, PABA), 4.60 (s,2H, 6-CH₂), 4.28 (m, 1H, Glu), 3.30-3.08 (m, 2H, cysteamine), 3.05 (m,6H, TEA), 2.37 (m, 2H, cysteamine), 2.10 (m, 4H, Glu), 1.20 (m, 9H,TEA). MS/ESI⁻ m/z 608.02 [M−H]⁻. It is worth noting that the isolationof 30 as its TEA⁺ or Na⁺ salt made it soluble in DMSO and/or water,which is an important requirement for its use in conjugation reactions.

EXAMPLE 3 Post Synthetic Conjugation of Enzymatic Nucleic Acid to FormNucleic Acid-Folate Conjugate (33) (FIG. 10)

Oligonucleotide synthesis, deprotection and purification was performedas described herein. 5′-Thiol-Modifier C6 (Glen Research, Sterling, Va.)was coupled as the last phosphoramidite to the 5′-end of a growingoligonucleotide chain. After cleavage from the solid support and basedeprotection, the disulfide modified enzymatic nucleic acid molecule 31(FIG. 10) was purified using ion exchange chromatography. The thiolgroup was unmasked by reduction with dithiothreitol (DTT) to afford 32which was purified by gel filtration and immediately conjugated with 30.The resulting conjugate 33 was separated from the excess folate by gelfiltration and then purified by RP HPLC using gradient of acetonitrilein 50 mM triethylammonium acetate (TEAA). Desalting was performed by RPHPLC. Reactions were conducted on 400 mg of disulfide modified enzymaticnucleic acid molecule 31 to afford 200-250 mg (50-60% yield) ofconjugate 33. MALDI TOF MS confirmed the structure: 13 [M−H]⁻ 12084.74(calc. 12083.82). An alternative approach to this synthesis is shown inFIG. 11.

As shown in Examples 2 and 3, a folate-cysteamine adduct can be preparedby a scaleable solution phase synthesis in a good overall yield.Disulfide conjugation of this novel targeting ligand to thethiol-modified oligonucleotide is suitable for the multi-gram scalesynthesis. The 9-atom spacer provides a useful spatial separationbetween folate and attached oligonucleotide cargo. Importantly,conjugation of folate to the oligonucleotide through a disulfide bondshould permit intermolecular separation which was suggested to berequired for the functional cytosolic entry of a protein drug.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein are exemplary and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art, which are encompassedwithin the spirit of the invention, are defined by the scope of theclaims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byvarious embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Other embodiments are within the following claims.

TABLE I Characteristics of naturally occurring ribozymes Group I IntronsSize: ~150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintenance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue- green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies[^(i),^(ii)]. Complete kinetic framework established for one ribozyme[^(iii),^(iv),^(v),^(vi)]. Studies of ribozyme folding and substratedocking underway [^(vii),^(viii),^(ix)]. Chemical modificationinvestigation of important residues well established [^(x),^(xi)]. Thesmall (4-6 nt) binding site may make this ribozyme too non-specific fortargeted RNA cleavage, however, the Tetrahymena group I intron has beenused to repair a “defective” β-galactosidase message by the ligation ofnew β-galactosidase sequences onto the defective message [^(xii)]. RNAseP RNA (M1 RNA) Size: ~290 to 400 nucleotides. RNA portion of aubiquitous ribonucleoprotein enzyme. Cleaves tRNA precursors to formmature tRNA [^(xiii)]. Reaction mechanism: possible attack by M²⁺-OH togenerate cleavage products with 3′-OH and 5′-phosphate. RNAse P is foundthroughout the prokaryotes and eukaryotes. The RNA subunit has beensequenced from bacteria, yeast, rodents, and primates. Recruitment ofendogenous RNAse P for therapeutic applications is possible throughhybridization of an External Guide Sequence (EGS) to the target RNA[^(xiv),^(xv)] Important phosphate and 2′ OH contacts recentlyidentified [^(xvi),^(xvii)] Group II Introns Size: >1000 nucleotides.Trans cleavage of target RNAs recently demonstrated [^(xviii),^(xix)].Sequence requirements not fully determined. Reaction mechanism: 2′-OH ofan internal adenosine generates cleavage products with 3′-OH and a“lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Only naturalribozyme with demonstrated participation in DNA cleavage [^(xx),^(xxi)]in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [^(xxii)].Important 2′ OH contacts beginning to be identified [^(xxiii)] Kineticframework under development [^(xxiv)] Neurospora VS RNA Size: ~144nucleotides. Trans cleavage of hairpin target RNAs recently demonstrated[^(xxv)]. Sequence requirements not fully determined. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. Binding sites andstructural requirements not fully determined. Only 1 known member ofthis class. Found in Neurospora VS RNA. Hammerhead Ribozyme (see textfor references) Size: ~13 to 40 nucleotides. Requires the targetsequence UH immediately 5′ of the cleavage site. Binds a variable numbernucleotides on both sides of the cleavage site. Reaction mechanism:attack by 2′-OH 5′ to the scissile bond to generate cleavage productswith 2′,3′-cyclic phosphate and 5′-OH ends. 14 known members of thisclass. Found in a number of plant pathogens (virusoids) that use RNA asthe infectious agent. Essential structural features largely defined,including 2 crystal structures [^(xxvi),^(xxvii)] Minimal ligationactivity demonstrated (for engineering through in vitro selection)[^(xxviii)] Complete kinetic framework established for two or moreribozymes [^(xxix)]. Chemical modification investigation of importantresidues well established [^(xxx)]. Hairpin Ribozyme Size: ~50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at the 5′-side of the cleavage siteand a variable number to the 3′- side of the cleavage site. Reactionmechanism: attack by 2′-OH 5′ to the scissile bond to generate cleavageproducts with 2′,3′-cyclic phosphate and 5′-OH ends. 3 known members ofthis class. Found in three plant pathogen (satellite RNAs of the tobaccoringspot virus, arabis mosaic virus and chicory yellow mottle virus)which uses RNA as the infectious agent. Essential structural featureslargely defined [^(xxxi),^(xxxii),^(xxxiii),^(xxxiv)] Ligation activity(in addition to cleavage activity) makes ribozyme amenable toengineering through in vitro selection [^(xxxv)] Complete kineticframework established for one ribozyme [^(xxxvi)]. Chemical modificationinvestigation of important residues begun [^(xxxvii),^(xxxviii)].Hepatitis Delta Virus (HDV) Ribozyme Size: ~60 nucleotides. Transcleavage of target RNAs demonstrated [^(xxxix)]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [^(xl)]. Reaction mechanism: attack by 2′-OH 5′ to thescissile bond to generate cleavage products with 2′,3′-cyclic phosphateand 5′-OH ends. Only 2 known members of this class. Found in human HDV.Circular form of HDV is active and shows increased nuclease stability[^(xli)] ^(i)Michel, Francois; Westhof, Eric. Slippery substrates. Nat.Struct. Biol. (1994), 1(1), 5-7. ^(ii)Lisacek, Frederique; Diaz,Yolande; Michel, Francois. Automatic identification of group I introncores in genomic DNA sequences. J. Mol. Biol. (1994), 235(4), 1206-17.^(iii)Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNA cleavage bythe Tetrahymena thermophila ribozyme. 1. Kinetic description of thereaction of an RNA substrate complementary to the active site.Biochemistry (1990), 29(44), 10159-71. ^(iv)Herschlag, Daniel; Cech,Thomas R.. Catalysis of RNA cleavage by the Tetrahymena thermophilaribozyme. 2. Kinetic description of the reaction of an RNA substratethat forms a mismatch at the active site. Biochemistry (1990), 29(44),10172-80. ^(v)Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies ofthe Tetrahymena Ribozyme Reveal an Unconventional Origin of an ApparentpKa. Biochemistry (1996), 35(5), 1560-70. ^(vi)Bevilacqua, Philip C.;Sugimoto, Naoki; Turner, Douglas H.. A mechanistic framework for thesecond step of splicing catalyzed by the Tetrahymena ribozyme.Biochemistry (1996), 35(2), 648-58. ^(vii)Li, Yi; Bevilacqua, Philip C.;Mathews, David; Turner, Douglas H.. Thermodynamic and activationparameters for binding of a pyrene-labeled substrate by the Tetrahymenaribozyme: docking is not diffusion-controlled and is driven by afavorable entropy change. Biochemistry (1995), 34(44), 14394-9.^(viii)Banerjee, Aloke Raj; Turner, Douglas H.. The time dependence ofchemical modification reveals slow steps in the folding of a group Iribozyme. Biochemistry (1995), 34(19), 6504-12. ^(ix)Zarrinkar, PatrickP.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guidefolding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5),854-8. ^(x)Strobel, Scott A.; Cech, Thomas R.. Minor groove recognitionof the conserved G. cntdot. U pair at the Tetrahymena ribozyme reactionsite. Science (Washington, D.C.) (1995), 267(5198), 675-9. ^(xi)Strobel,Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G. cntdot. UPair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to5′-Splice Site Selection and Transition State Stabilization.Biochemistry (1996), 35(4), 1201-11. ^(xii)Sullenger, Bruce A.; Cech,Thomas R.. Ribozyme-mediated repair of defective mRNA by targetedtrans-splicing. Nature (London) (1994), 371(6498), 619-22.^(xiii)Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol. Chem., 247,5243-5251 (1972). ^(xiv)Forster, Anthony C.; Altman, Sidney. Externalguide sequences for an RNA enzyme. Science (Washington, D.C., 1883-)(1990), 249(4970), 783-6. ^(xv)Yuan, Y.; Hwang, E. S.; Altman, S.Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA(1992) 89, 8006-10. ^(xvi)Harris, Michael E.; Pace, Norman R..Identification of phosphates involved in catalysis by the ribozyme RNaseP RNA. RNA (1995), 1(2), 210-18. ^(xvii)Pan, Tao; Loria, Andrew; Zhong,Kun. Probing of tertiary interactions in RNA: 2′-hydroxyl-base contactsbetween the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A.(1995), 92(26), 12510-14. ^(xviii)Pyle, Anna Marie; Green, Justin B..Building a Kinetic Framework for Group II Intron Ribozyme Activity:Quantitation of Interdomain Binding and Reaction Rate. Biochemistry(1994), 33(9), 2716-25. ^(xix)Michels, William J. Jr.; Pyle, Anna Marie.Conversion of a Group II Intron into a New Multiple- Turnover Ribozymethat Selectively Cleaves Oligonucleotides: Elucidation of ReactionMechanism and Structure/Function Relationships. Biochemistry (1995),34(9), 2965-77. ^(xx)Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang,Jian; Perlman, Philip S.; Lambowitz, Alan M..A group II intron RNA is acatalytic component of a DNA endonuclease involved in intron mobility.Cell (Cambridge, Mass.) (1995), 83(4), 529-38. ^(xxi)Griffin, Edmund A.,Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group IIintron ribozymes that cleave DNA and RNA linkages with similarefficiency, and lack contacts with substrate 2′-hydroxyl groups. Chem.Biol. (1995), 2(11), 761-70. ^(xxii)Michel, Francois; Ferat, Jean Luc.Structure and activities of group II introns. Annu. Rev. Biochem.(1995), 64, 435-61. ^(xxiii)Abramovitz, Dana L.; Friedman, Richard A.;Pyle, Anna Marie. Catalytic role of 2′-hydroxyl groups within a group IIintron active site. Science (Washington, D.C.) (1996), 271(5254),1410-13. ^(xxiv)Daniels, Danette L.; Michels, William J., Jr.; Pyle,Anna Marie. Two competing pathways for self-splicing by group IIintrons: a quantitative analysis of in vitro reaction rates andproducts. J. Mol. Biol. (1996), 256(1), 31-49. ^(xxv)Guo, Hans C. T.;Collins, Richard A.. Efficient trans-cleavage of a stem-loop RNAsubstrate by a ribozyme derived from Neurospora VS RNA. EMBO J. (1995),14(2), 368-76. ^(xxvi)Scott, W. G., Finch, J. T., Aaron, K. The crystalstructure of an all RNA hammerhead ribozyme:Aproposed mechanism for RNAcatalytic cleavage. Cell, (1995), 81, 991-1002. ^(xxvii)McKay, Structureand function of the hammerhead ribozyme: an unfinished story. RNA,(1996), 2, 395-403. ^(xxviii)Long, D., Uhlenbeck, O., Hertel, K.Ligation with hammerhead ribozymes. U.S. Pat. No. 5,633,133.^(xxix)Hertel, K. J., Herschlag, D., Uhlenbeck, O. A kinetic andthermodynamic framework for the hammerhead ribozyme reaction.Biochemistry, (1994) 33, 3374-3385. Beigelman, L., et al., Chemicalmodifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270,25702-25708. ^(xxx)Beigelman, L., et al., Chemical modifications ofhammerhead ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.^(xxxi)Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip.‘Hairpin’ catalytic RNA model: evidence for helixes and sequencerequirement for substrate RNA. Nucleic Acids Res. (1990), 18(2),299-304. ^(xxxii)Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke,John M.. Novel guanosine requirement for catalysis by the hairpinribozyme. Nature (London) (1991), 354(6351), 320-2.^(xxxiii)Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat M.;Butcher, Samuel E.; Burke, John M.. Essential nucleotide sequences andsecondary structure elements of the hairpin ribozyme. EMBO J. (1993),12(6), 2567-73. ^(xxxiv)Joseph, Simpson; Berzal-Herranz, Alfredo;Chowrira, Bharat M.; Butcher, Samuel E.. Substrate selection rules forthe hairpin ribozyme determined by in vitro selection, mutation, andanalysis of mismatched substrates. Genes Dev. (1993), 7(1), 130-8.^(xxxv)Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M.. Invitro selection of active hairpin ribozymes by sequential RNA-catalyzedcleavage and ligation reactions. Genes Dev. (1992), 6(1), 129-34.^(xxxvi)Hegg, Lisa A.; Fedor, Martha J.. Kinetics and Thermodynamics ofIntermolecular Catalysis by Hairpin Ribozymes. Biochemistry (1995),34(48), 15813-28. ^(xxxvii)Grasby, Jane A.; Mersmann, Karin; Singh,Mohinder; Gait, Michael J.. Purine Functional Groups in EssentialResidues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA.Biochemistry (1995), 34(12), 4068-76. ^(xxxviii)Schmidt, Sabine;Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim; Sorensen, UlrikS.; Gait, Michael J.. Base and sugar requirements for RNA cleavage ofessential nucleoside residues in internal loop B of the hairpinribozyme: implications for secondary structure. Nucleic Acids Res.(1996), 24(4), 573-81. ^(xxxix)Perrotta, Anne T.; Been, Michael D..Cleavage of oligoribonucleotides by a ribozyme derived from thehepatitis. delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.^(xl)Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structurerequired for efficient self-cleavage of hepatitis delta virus RNA.Nature (London) (1991), 350(6317), 434-6. ^(xli)Puttaraju, M.; Perrotta,Anne T.; Been, Michael D.. A circular trans-acting hepatitis delta virusribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

TABLE II Wait Wait Time* Wait Reagent Equivalents Amount Time* DNA2′-O-methyl Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents:DNA/ Amount: DNA/2′-O- Wait Time* Wait Time* WaitTime* Reagent 2′-O-methyl/Ribo methyl/Ribo DNA 2′-O-methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA *Wait time does not includecontact time during delivery.

1. A compound having the formula VI:

wherein each R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, R₂ isa nucleic acid that is double stranded, each “n” is independently aninteger from 0 to about 200, and L is :

each R₃ and R₄ is independently hydrogen, alkyl, substituted alkyl,aryl, substituted aryl, or a protecting group and R₁₃ is an amino acidside chain.
 2. The compound of claim 1, wherein R₃, R₄, R₅, R₆ and R₇are hydrogen.
 3. The compound of claim 1 wherein the nucleic acid is anenzymatic nucleic acid.